Note: Descriptions are shown in the official language in which they were submitted.
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ANTI-VIRAL COMPOUNDS, PHARMACEUTICAL COMPOSITIONS,
AND METHODS OF USE THEREOF
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Patent
Application No.
61/991,418 filed on May 9, 2014, and U.S. Provisional Application No.
62/177,900, filed
on March 25, 2015, each of which is incorporated by reference herein in their
entirety.
FIELD OF THE DISCLOSURE
[0002] Compounds, pharmaceutical compositions, and methods disclosed herein
are
useful for treating viral infection, including RNA viral infection, in
subjects.
BACKGROUND OF THE DISCLOSURE
[0003] As a group, RNA viruses represent an enormous public health problem in
the
U.S. and worldwide. Well-known RNA viruses include influenza virus (including
the
avian and swine isolates; also referred to herein as flu), Hepatitis C virus
(HCV),
Dengue virus (DNV), West Nile virus (WNV), SARS-coronavirus (SARS), MERS-
coronavirus (MERS), respiratory syncytial virus (RSV), and human
immunodeficiency
virus (HIV). These viruses are responsible for pandemic outbreaks and threats
to public
health that have occurred throughout history. Flaviviruses, Henipaviruses,
Filoviruses,
and Arenaviruses are among emerging RNA viruses that pose significant public
health
and biodefense threats. These viruses collectively place hundreds of millions
of people
at risk of infection throughout the world. Many of the emerging RNA viruses
cause viral
hemorrhagic fever and can result in significant morbidity and mortality.
Dengue virus
(DNV) and West Nile virus (WNV) are both Flaviviruses (positive strand RNA
virus) and
Arboviruses, transmitted through mosquitoes; thus these viruses represent a
potent
potential biological threat through their ability to transmit readily among
insects or
animals and humans, high infectivity, and their potential to be weaponized in
bioterror
events.
[0004] At least 4 subtypes of Ebola virus (EV) are infectious to humans
(Zaire, Sudan,
Bundibugyo, and Cote d'Ivoire). EV outbreaks have been described in Africa
with a
fatality rate of up to 90%. Feldmann, H., et al. (2011) Lancet 49, 1-14. Cases
of EV
infection have been reported in other countries including, very recently, the
United
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States. The natural host for EV is not defined, but nonhuman primates (NHP)
are
susceptible. EV is a negative-strand RNA virus of the Filoviridae and can be
spread
effectively from person-to-person.
[0005] Seasonal flu infects 5 ¨ 20% of the population annually, resulting in
200,000
hospitalizations and 36,000 deaths. Influenza can precipitate viral or
secondary
bacterial pneumonia, and complicated disease in those at the extremes of age
or with
weakened immune systems. Coronaviruses are common throughout the world and
typically cause mild to moderate respiratory illness, although certain
coronaviruses
cause severe respiratory illness and death. A 2003 multi-country outbreak of
SARS-
coronavirus infection resulted in approximately 8,000 infections and nearly
800 deaths.
Recently there have been reported cases of Middle East Respiratory Syndrome
caused
by MERS-coronavirus.
[0006] More than 170 million people worldwide are infected by HCV, and 130
million of
these are chronic carriers at risk of developing chronic liver diseases
(cirrhosis,
carcinoma, and liver failure). As such, HCV is responsible for two thirds of
all liver
transplants in the developed world. Recent studies show that the death rate
from HCV
infection is rising due to the increasing age of chronically infected
patients.
[0007] DNV is the most prevalent flavivirus in humans, is endemic in most
tropical and
subtropical countries, and is currently emerging elsewhere including the U.S.
and
across the Pacific Islands. DNV circulates as 4 serotypes (DNV1 ¨ 4) and
following a
first infection, re-infection can lead to fatal hemorrhagic fever and shock
syndrome.
Infection is believed to provide life-long immunity against reinfection by the
same
serotype, but not against other serotypes. Epidemic outbreaks have been
reported in
many countries throughout Latin America, South-East Asia, and the Western
Pacific
Regions. It is estimated that between 50 and 100 million cases of Dengue fever
occur
globally each year, Dengue Hemorrhagic Fever and Dengue Shock Syndrome
represent severe forms of the disease. Currently there is no specific
antiviral therapy to
treat DNV infection and no approved vaccine.
[0008] WNV is a related flavivirus that is endemic in regions of Africa and
Asia, but is
now emerging in the Western hemisphere. WNV is neuroinvasive to cause serious
encephalitis disease and is lethal in about 6% of cases. Neuroinvasive WNV can
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present as meningitis, encephalitis or less frequently a flaccid paralysis
referred to as
poliomyelitis. WNV was largely absent from North America prior to 1999, but
reemerged
on the continent following an isolated outbreak of encephalitis in New York.
In the
subsequent 7 years, WNV infection spread throughout the 48 contiguous United
States,
and current estimates suggest as many as 2 ¨ 3 million Americans have been
infected.
Over the past 20 years, outbreaks have been reported in parts of Europe, North
Africa,
the Middle East, and North America. Currently there is no specific antiviral
therapy to
treat WNV infection and no approved vaccine.
[0009] Among the RNA viruses listed, very few vaccines are currently approved
for
clinical use. One such vaccine exists for influenza virus, which must be
revised and
administered annually. Accordingly, drug therapy is essential to mitigate the
significant
morbidity and mortality associated with these viruses. Unfortunately, the
number of
antiviral drugs is limited, many are poorly effective, and nearly all are
plagued by the
rapid evolution of viral resistance and a limited spectrum of action.
Ribavirin, a guanine
nucleoside analog, has been studied in clinical trials of diverse RNA virus
infections and
is likely the most broadly acting antiviral agent available. Ribavirin is
approved to treat
Hepatitis C virus (HCV) and respiratory syncytial virus (RSV) infection, and
Lassa virus
related mortality was shown to be reduced with intravenous ribavirin
treatment.
However, it is weakly effective as a single agent and has significant
hematologic
toxicity. Both classes of acute influenza antivirals, adamantanes and
neuraminidase
inhibitors, are only effective within the first 48 hours after infection,
thereby limiting the
window of opportunity for treatment. High resistance to adamantanes already
restricts
their use, and massive stockpiling of neuraminidase inhibitors will eventually
lead to
overuse and the emergence of resistant strains of influenza.
[0010] Based on the foregoing, there is an immense and unmet need for
effective
treatments against viral infections. Most drug development efforts against
viruses target
viral proteins. RNA viruses have small genomes, with many encoding less than a
dozen
proteins, resulting in a very limited number of viral targets for new drugs.
This is a large
part of the reason that current drugs are narrow in spectrum and subject to
the
emergence of viral resistance. However, there is benefit to discovery of new
viral
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targets for inhibition. Alternatively, direct-acting antiviral therapy can
work to counteract
any infection mechanisms such as viral entry into a host cell.
[0011] New antiviral therapy can act directly against viruses.
In particular, new
antiviral therapy can exploit the fact that these viruses are susceptible to
control by
innate intracellular immune defenses that function to suppress virus
replication and
spread. Compounds that act on cellular targets are likely to be more
effective, be less
susceptible to the emergence of viral resistance, cause fewer side effects,
and be
effective against a range of different viruses. An effective broad-spectrum
antiviral,
whether used on its own or in combination with other therapies, would be an
enormous
benefit to current clinical practice. While interferon is in principal host-
mediated and
broad spectrum, many viruses have evolved the ability to disrupt interferon
signaling
downstream of drug action at the receptor. An important criterion is the
development of
drugs that activate innate immune signaling below specific virus
countermeasures and
are a unique addition to conventional antiviral compounds in development or on
the
market. As one such innate immune antiviral response, the RIG-I-like receptor
(RLR)
pathway of innate antiviral immunity can impose a potent blockade to RNA virus
infection through the actions of a variety of antiviral defense genes.
SUMMARY OF THE DISCLOSURE
[0012] The compounds, pharmaceutical compositions, and methods disclosed
herein
shift the focus of viral drug development away from the targeting of viral
proteins to the
targeting and enhancing of the host's innate antiviral immune response. The
present
disclosure relates to compounds, pharmaceutical compositions including the
compounds, and associated methods of use to treat viral infection, including
RNA viral
infection. In certain embodiments, the compounds modulate the RIG-I pathway.
[0013] Embodiments of the present disclosure can provide compounds represented
by
the formula
1
Y
y?,.:1
-:- .... W,..,\\
L V¨Q R1
Y.`;=,. - hr
4
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wherein L is NR2, 0, S, C(=O)N, CR2R3CR2R3, CR2R3NR2, CR4=CR4, CR2R30,
CR2R3S, NR2CR2R3, NR2C(=0), NS(0)t, OCR2R3, SCR2R3;
V is (CR2R3)u, C(=0)CR2R3, CR2R30, CR2R3OCR2R3,CR4=CR4, CE-C, C(=NR2),
or C(=0);
Q is NR2, 0, S(0)t, or a bond;
t = 0, 1, 2; u= 0-3;
wherein a dashed line indicates the presence or absence of a bond;
R1 is Ra, 0R2, or NR2R3;
each Ra is independently H, optionally substituted hydrocarbyl, optionally
substituted aryl, optionally substituted heteroaryl;
R2 and R3 are each independently Ra, C(=0)Ra, SO2Ra, or R2 and R3 form an
optionally substituted carbocyclic, heterocarbocyclic, aryl, or heteroaryl
ring;
each R4 is independently R2, ORa, C(=0)Ra, C(=0)NR2R3, NR2R3, NRb(=0)Ra,
SR, SORa, SO2Ra, SO2NHRa, SO2NR2R3, NCORa, halogen, trihalomethyl, CN, S=0,
nitro, or two R4 groups form an optionally substituted carbocyclic,
heterocarbocyclic,
aryl, or heteroaryl ring;
W and X are each independently N, NRa, NR5, 0, S, CR2R4 or CR4;
R5 is Ra, C(=0)Ra, SO2Ra, or is not present;
yi, Y2, Y3 and r are each independently CR4 or N; and
NR2R3 may form an optionally substituted heterocylic or heteroaryl ring
including
pyrrolidine, piperidine, morpholine, and piperazine.
[0014] In some embodiments, compounds can be represented by the formula
(R4)n
111
0 _________________________________________
N
H N
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wherein R4 is Rd, SO2Rd, C(=0)Rd, NH C(=0)Rd, Re, ORc, or CF3, wherein RC is H
or Ci-
Cio Rd is unsubstituted heterocyclic or unsubstituted carbocyclic,
and Re is
substituted heteroaryl or substituted phenyl; and
n is 1 or 2.
[0015] Some embodiments of the present disclosure can include a pharmaceutical
composition including any of the compounds as described herein.
[0016] In addition, embodiments of the present disclosure can include methods
of
treating a viral infection in a subject including administering to the subject
a
therapeutically effective dose of a pharmaceutical composition as described
herein
thereby treating the viral infection in the subject.
[0017] Further, embodiments of the methods of the present disclosure can
include
administering any of the pharmaceutical compositions described herein as an
adjuvant
for a prophylactic or therapeutic vaccine.
[0018] Embodiments of the present disclosure include methods of modulating the
innate immune response in a eukaryotic cell, including administering to the
cell any of
the compounds as described herein. In some embodiments the cell is in vivo. In
other
embodiments the cell is in vitro.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figures 1A-1D show in vitro biological activity. In Figure 1A, the high-
throughput
screen "hit" compound, compound 1 of Table 1, was validated by demonstrating
dose-
dependent induction of the IFN6-luciferase (IFNp-Luc, left), ISG56-luciferase
(ISG56-
LUC, center), and the ISG54-luciferase (ISG54-LUC, right) reporter genes. RLU
=
relative luciferase units. Figure 1B confirms the specificity of compound 1,
10 pM of
which (compound 1) does not induce the non-specific 6-actin promoter relative
to
vehicle control (DMSO), and in contrast to equivalent dose of a positive
control
compound (CPD X). Figure 1C shows HeLa cells treated with increasing amounts
of
compound 1 showed dose-dependent increase in interferon regulatory factor
(IRF)-3
translocation to the nucleus, quantified by nuclear intensity minus
cytoplasmic intensity
("normalized nuclear intensity"). Figure 1D shows HeLa cells treated with
increasing
amounts of compound 1 showed dose-dependent increase in NFKB translocation,
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quantified by nuclear intensity minus cytoplasmic intensity. "SeV" refers to
Sendai virus
infection, the positive control.
[0020] Figures 2A-2C show induction of gene expression by compound 1 and 2 of
Table 1. Figure 2A shows gene expression levels of IFIT2 (left) and OAS1
(right) in
HeLa cells over time from 4-24 hours post treatment with 1 OpM compound 1
(grey;
OAS1 only) or 10pM compound 2 (black; IFIT2 and OAS1 both shown). Figure 2B
shows gene expression levels of IFIT2 in PH5CH8 cells (solid color bars) and
HeLa
cells (black checked bars) treated with 1 OpM compound 1 (CPD 1) or compound 2
(CPD 2). Figure 2C shows gene expression levels of IFIT2 (left), OAS1
(center), and
MxA (right) in primary HUVEC cells that were treated with 11.iM compound 1
(CPD 1) or
11.IM compound 2 (CPD 2).
[0021] Figures 3A-3B show induction of gene expression by compound 3 and
compound 7 of Table 1. Figure 3A shows IFIT2 gene expression was induced by
5pM
compound 3 or compound 7. Figure 3B shows compound 3 induced innate immune
gene expression in mouse macrophage cells.
[0022] Figure 4 shows induction of the chemokines IL-8, MCP-1, MIP-1a, and MIP-
113
by dendritic cells treated with compound 1 of Table 1 (concentrations shown in
pM).
LPS is shown as a positive control inducer of chemokine expression.
[0023] Figure 5 shows results of experiments performed using the protocol of
Example
8, demonstrating the antiviral activity of select compounds of Figure 5 that
demonstrated antiviral activity against RSV. +++ = greater than 70% inhibition
of
infection, ++ = greater than 50% inhibition, + = greater than 30% inhibition, -
= less than
30% inhibition.
[0024] Figure 6 shows antiviral activity of example compounds against
Influenza A
virus Udorn/72. Treatment of HEK293 cells with increasing concentrations of
compound
3, compound 7, compound 9, and compound 10 of Table 1 resulted in dose-
dependent
inhibition of virus infection (shown as A untreated negative control).
Calculated IC50
values are shown.
[0025] Figure 7 shows the antiviral activity of compound 5 and compound 20 of
Table
1 against Dengue virus (DNV) type 2. Treatment with increasing amounts of the
compounds showed dose-dependent decrease in infection by virus.
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[0026] Figure 8 shows antiviral activity of example compounds against DNV type
2.
Treatment of Huh 7 cells with increasing concentrations of compound 8,
compound 3,
compound 5, compound 6, compound 7, compound 9, and compound 10 of Table 1
resulted in dose-dependent inhibition of virus infection (shown as % untreated
negative
control). Calculated IC50 values are shown.
[0027] Figure 9 shows antiviral activity of example compounds against human
coronavirus 0C43. Treatment with increasing concentrations of compound 3,
compound
5, compound 6, and compound 7 of Table 1 resulted in dose-dependent inhibition
of
virus infection (shown as % untreated negative control). Calculated IC50
values are
shown.
[0028] Figure 10 shows results from exploratory pharmacokinetic (PK) studies.
Figure
10A,administration of compound 3 of Table 1 via oral (PO) or intravenous (IV)
route
resulted in detectable levels of compound in serum samples obtained up to 250
minutes
post treatment. Figure 10B, at 4 hours post treatment of compound 3 and
compound 7
of Table 1, there was detectable compound in lung tissue.
[0029] Figure 11A-11C show a study performed using the mouse hepatitis virus
type 1
(MHV-1) coronavirus model. Treatment with compound 3 of Table 1 resulted in
decreased pathological symptoms including weight loss Figure 11A and increased
survival 11B after lethal challenge with MHV-1. Figure 11C virus was decreased
in the
lung of animals treated with compound 3.
[0030] Figure 12 shows the in vitro activity of compound 12 of Table 1 of the
disclosure
against EBOV at 5 pM, showing greater than a 2 log reduction in EBOV titer in
vitro.
[0031] Figure 13 shows the dose response activity of compound 8 of Table 1
against
DENV-2, as FFU/ml.
DETAILED DESCRIPTION
[0032] The present disclosure provides compounds, pharmaceutical compositions,
and
methods that shift the focus of viral treatments away from the targeting of
viral proteins
to targeting and enhancing the host (subject's) innate antiviral response.
Such
compounds, pharmaceutical compositions, and methods are likely to be more
effective,
less susceptible to the emergence of viral resistance, cause fewer side
effects, and be
effective against a range of different viruses.
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[0033] The retinoic acid-inducible gene 1 (RIG-I) pathway is intimately
involved in
regulating the innate immune response to virus infections including RNA virus
infections. RIG-I is a cytosolic pathogen recognition receptor that is
essential for
triggering immunity to a wide range of RNA viruses. RIG-I is a double-stranded
RNA
helicase that binds to motifs within the RNA virus genome characterized by
homopolymeric stretches of uridine or polymeric UM motifs. Binding to RNA
induces a
conformation change that relieves RIG-I signaling repression by an autologous
repressor domain, thus allowing RIG-I to signal downstream through its tandem
caspase activation and recruitment domains (CARDS). RIG-I signaling is
dependent
upon its NTPase activity, but does not require the helicase domain. RIG-I
signaling is
silent in resting cells, and the repressor domain serves as the on-off switch
that governs
signaling in response to virus infection.
[0034] Without being bound by a theory or particular mechanism of action, RIG-
I
signaling is transduced through IPS-1 (also known as Cardif, MAVs, and VISA),
an
essential adaptor protein that resides in the outer mitochondrial membrane.
IPS-1
recruits a macromolecular signaling complex that stimulates the downstream
activation
of IRF-3, a transcription factor that induces the expression of type I
interferons (IFNs)
and virus-responsive genes that control infection. Compounds that trigger RIG-
I
signaling directly or through modulation of RIG-I pathway components,
including IRF-3,
present attractive therapeutic applications as anfivirals or immune
modulators.
[0035] In certain embodiments, a high-throughput screening approach was used
to
identify compounds that modulate the RIG-I pathway. In particular embodiments,
validated RIG-I agonist lead compounds were demonstrated to specifically
activate IRF-
3. In additional embodiments, the compounds have one or more of the following
advantages: induce expression of interferon-stimulated genes (ISGs low
cytotoxicity in
cell-based assays, suitable for analog development and SAR studies, drug-like
physiochemical properties, and/or antiviral activity against viruses including
Dengue
virus (DNV), human coronavirus (SARS and MERS-like pathogen), influenza A
virus,
respiratory syncytial virus (RSV), and/or Hepatitis C virus (HCV). In
additional
embodiments, the compounds exhibit antiviral activity against dsDNA viruses
including
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human cytomegalovirus. In certain embodiments, the compounds exhibit all of
these
characteristics.
[0036] The disclosed compounds represent a new class of antiviral
therapeutics.
Although the disclosure is not bound by a specific mechanism of action of the
compounds in vivo, the compounds are selected for their modulation of innate
immune
antiviral responses. In certain embodiments, the modulation is activation of
the RIG-I
pathway. Compounds, pharmaceutical compositions, and methods disclosed herein
function to decrease one or more of: viral protein, viral RNA, and infectious
virus in
laboratory models of viral infection.
[0037] The disclosure herein relates to a class of compounds represented by
Formula
1:
2-2 "s=-.
Y
3 s __ = __
-L V -Q ____ R1
/
Formula 1
wherein L can be NR2, 0, S, C(=O)N, CR2R3CR2R3, CR2R3NR2, CR4=CR4, CR2R30,
CR2R3S, NR2CR2R3, NR2C(=0), NS(0)t, OCR2R3, SCR2R3;
V is (CR2R3)u, C(=0)CR2R3, CR2R30, CR2R3OCR2R3,CR4=CR4, GEC, C(=NR2), or
C(=0);
Q can be NR2, 0, S(0)t, or a bond;
t = 0, 1, 2; u= 0-3;
wherein a dashed line indicates the presence or absence of a bond; R1 can be
Ra, 0R2
or NR2R3; each Ra can independently be H, optionally substituted hydrocarbyl,
optionally substituted aryl, optionally substituted heteroaryl; R2 and R3 can
each
independently be Ra, C(=0)Ra, or SO2Ra, R2 and R3 can form an optionally
substituted
carbocyclic, heterocarbocyclic, aryl or heteroaryl ring; each R4 can
independently be
R2, ORa, C(=0)Ra, C(=0)NR2R3, NR2R3, NRb(=0)Ra, SR, SORa, SO2Ra, SO2NHRa,
SO2NR2R3, NCORa, halogen, trihalomethyl, CN, S=0, or nitro; W and X can each
independently be N, NRa, NR5, 0, S, CR2R4 or CR4; R5 can be Ra, C(=0)Ra,
SO2Ra, or
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is not present; Y1, Y2, Y3 and Y4can each independently be CR4 or N; and NR2R3
can
form an optionally substituted heterocylic or heteroaryl ring including, but
not limited to,
pyrrolidine, piperidine, morpholine, and piperazine.
[0038] In an embodiment, with respect to Formula 1, Y1 can be CR4 or N. For
example, Y1 can be CR4.
[0039] In an embodiment, with respect to Formula 1, Y2 can be CR4 or N. For
example, Y2 can be CR4.
[0040] In an embodiment, with respect to Formula 1, Y2 can be CR4 or N. For
example, Y3 can be CR4.
[0041] In an embodiment, with respect to Formula 1, Y1 and Y2 can both be CR4,
and
in some instances, Y1 and Y2 can form a fused heterocyclic ring optionally
substituted
by R6 as shown below:
R6
----: N
-----
S
4
i
wherein R6 can be H, CH3, CH2CH3, Cl, Br, OH, OCH3, SCH3, NH2, NHCH3, N(CH3)2,
SO2NH2, morpholino, CH2C---CH, or NO2. For example, R6 can be H or CH3.
[0042] In an embodiment, with respect to Formula 1, Y3 and Y4 can be CR4 or N.
For
example, Y3 and Y4 can be CR4.
[0043] In an embodiment, with respect to Formula 1, )11, y2, y3 and Y4 can be
CR4. In
some instances, y1, y2, Y3 and Y4 can be CH. In some instances, Y1 and Y2 can
form a
fused heterocyclic ring optionally substituted by R6, as shown above, and Y3
and Y4 can
be CH.
[0044] The disclosure also relates to a class of compounds represented by
Formula
1A.
11
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R4
R4
-VV\
11/¨R1
R4 N
R4
Formula 1A
wherein W can be 0 or S; and R1 can be Ra, 0R2,or NR2R3. R1, R2, R3, R4, Ra,
and V
and W, can be as defined above with respect to Formula 1. In an embodiment,
each Ra
can independently be H, optionally substituted hydrocarbyl, optionally
substituted aryl,
optionally substituted heteroaryl; R2 and R3 can each independently be Ra,
CORa, or
S02R2; each R4 can independently be R2, ORa, NR2R3, SRa, SORa, SO2Ra, SO2NHRa,
NCORa, C(=0)Ra, CONR2R3, halogen, trihalomethyl, CN, S=0, or nitro; V can be
CR2R3, C(=0), C(=0)CR2R3, or C(=N)R2; and W can be 0 or S. In some instances,
V
can be C=0, and R1 can be an optionally substituted aryl, or an optionally
substituted
heteroaryl. For example, R1 can be optionally substituted phenyl, or R1 can be
optionally
substituted naphthyl.
[0045] The disclosure also relates to a class of compounds represented by
Formula
1 B.
R6
N
k
¨N __________________________________________ V ___ R
R4 N
4
Formula 1B
wherein W can be 0 or S; and R1 can be Ra or NR2R3. R1, R2, R3, R4, R6, rc
r%a, and V are
as defined above with respect to Formula 1.
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In some instances, V can be C=0, and R1 can be an optionally substituted aryl,
or an
optionally substituted heteroaryl. For example, R1 can be optionally
substituted phenyl,
or R1 can be optionally substituted naphthyl.
[0046] The disclosure also relates to a class of compounds represented by
Formula
1C.
R4
4
N __________________________________________ V ¨ R
4 \Ra
4
Formula 1C
wherein W can be 0 or S; and R1 can be Ra, 0R2, or NR2R3. R17 R2, R3, R4, Ra,
and V
and W, are as defined above with respect to Formula 1. In an embodiment, each
Ra can
independently be H, optionally substituted hydrocarbyl, optionally substituted
aryl,
optionally substituted heteroaryl; R2 and R3 can each independently be Ra,
C(=0)Ra, or
SO2Ra; each R4 can independently be R2, ORa, NR2R3, SRa, SORa, SO2Ra, SO2NHRa,
NCORa, C(=0)Ra, CONR2R3, halogen, trihalomethyl, CN, S=0, or nitro; V can be
CR2R3, C(=0), C(=0)CR2R3, or C(=N)R2; and W can be 0 or S. In some instances,
V
can be C=0, and R1 can be an optionally substituted aryl, or an optionally
substituted
heteroaryl. For example, R1 can be optionally substituted phenyl, or R1 can be
optionally
substituted naphthyl.
[0047] A class of compounds of interest can include compounds of Formula 1A,
1B, or
1C wherein R4 can be H; and V can be C=0. Some embodiments can include
compounds having Formula 1A, Formula, 1B, or Formula 1C wherein R1 is
optionally
substituted phenyl or optionally substituted naphthyl. Various embodiments can
include
compounds having Formula 1A, Formula, 1B, or Formula 1C wherein W is S and X
is N.
Additional embodiments can include compounds having Formula 1A, Formula, 18,
or
Formula 1C wherein W is 0 and X is N.
[0048] The disclosure also includes a class of compounds represented by any
one of
Formulas 2-11.
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R4
4
Y-R1
=
4
R= = === ==
\=
Ft. =
R4
FOrniula 2
4
R...y= = = = .
/==;==
5.
4: = .= ' =:= . = =
R.' ====:
Fporiula 3
P...
= =
R
Formula .4
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6
R
8: 1
\
),---------N
...... ././ \ 5
R4.---'"' =:. ,,,..':.''''. Kit R
R4
Fort-hula 5
0
........................................................ /
........................................................ ,
.. ______ ,. __
C\-,J: = ___ Ni .
Formula. 6
b
dy. .,,,/\c:
=, N
H
N.,,, =... s = __ ("II/ ':¨
) ..,........õ,. ,
Fo.mitila 7
/ \ .
':'. ...."':'' '. = -,:f41'
Formola 8
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F
I
F.- __ I F
i 0 r .. -Nµ
µ.8 \
\\ .................................... . / \ ____
.,,,,,,,,.. \ .,,,,,=-= S / /1/ N\
\\
'."'=,.,-...,/,.,-----.--1.1/1 \. ___ /
Formula 9
/-=
ii
ii
0 ,i----<,1 `
113C \- .. ¨\\4/ \ .. . __ -/
\ / \ _,/
}-13Cre")\ iii __ .N z
CH3 ,.._ ,,,,,,_ ,t,, H
Formula 10
/7r.---
0 _____________________________________________________ ii
H/
, \ ¨ - /
--
/
0I
.-- ,.,...,,--:::-------...N.\) N
// H
Formula 11
.1
1. ------- \
/.)
=:;,,,,,,,,.õ.i4s it .\
.17 7. 67
16
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Formula 12
[0049] Embodiments can include compounds having Formula 4 wherein R1 can be a
phenyl group substituted by at least one halogen, a phenyl group substituted
by NR2R3,
a phenyl group substituted by SO2NR2R3, CR2R3ORd, an unsubstituted naphthyl
group,
a naphthyl group substituted by 0(CR2R3)n Rd, NRa(CR2R3)nRd, NRa(CR2R3)nNR2R3,
a
two membered ring structure including a pyridynyl group and a phenyl group, or
a two
membered ring structure including a phenyl group and a dioxolanyl group; each
Ra can
independently be H or optionally substituted hydrocarbyl (Ci-Cio); R2 and R3
can each
independently be Ra, CORa, (CHOnO, or SO2Ra; each R4 can independently be Ra;
Rd
can be phenyl or morpholino; R5 can be H or CH3; R6 can be H or CH3; and
wherein n
can be 1, 2, 3, or 4.
[0050] Particular embodiments having Formula 4 can be represented by the
compounds
CH3
)----:--N
S 0
0 S Br
/ N----j
>---
H N
I
H
H
,
CH3
\1.----------= N
S 0
>----- N.-----
I I
H N H "=-.....õ?..,C1
H
,
17
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C H 3
)":7"-------- N
S 0
\
H 0 N/7¨ Nri 11. illi
0
1/49
H// *"-.... ipti ------
\ ,
H
s 0
40 1 s > I C I
/ ________________________ N
0
H N H -,....õ..,
H
Ci
H
\)"----:-- N
0
S \
H N H
0
H
0 ____________________________________________ /
,
18
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CH3
>'----- N
S 0
S Br
H N 1
CI-1
H ,
H
\\)-------- N
S 0
I / ______________________ N 41111 11
1
H,
CH3
)----74--- N
S 0
0
1 i 1
H N H H
,
19
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H
.------- N
--.----
H
Olio
H H
H
H
----- N
i)----
S '0
N
H ,
Ls...õ.0
411
, or
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H3C
HN
3 = -
S N
[0051] Embodiments can include compounds having Formula 5 wherein R1 can be a
phenyl group substituted by at least one halogen, a phenyl group substituted
by NR2R3,
a phenyl group substituted by SO2Rd, a naphthyl group optionally substituted
by
0(CR2R3)nRd, or an unsubstituted naphthyl group, each Ra can independently be
H or
optionally substituted Ci-Cio hydrocarbyl; R2, R3 and each R4 can
independently be Ra,
Rd can optionally be substituted phenyl or optionally substituted morpholino;
R6 can be
H or CH3; R6 can be H or CH3, and wherein n can be 1, 2, 3, or 4.
[0052] Particular embodiments having Formula 4 can be represented by the
compounds
H3C, N
411
\\ Br
0
N
0
'
\\\.,
21
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,--õ
.õ..õ...-- -...õ....,..::,..õ
H
,.. N 0 .,.. ..., N = .....--
ti:;---- t \ ___________________________________ / / ---y,"'8r
,
.,, \S I i
1 4,/ \ \ = I
S- di/ \\,,_..,N 0
--.....õ ,
. s
\ i
\ ___________ /
,
õ....,...
.......--- --:::...-....)
H i
== 1
H3C ......_...N /0,, ...,-N,,,
---es:=---- \ y'' ,....... ........ a
\ 1 i
S =-=., i'll Vs)......----= N 0
\ .. ¨ i ,
0H3
1
....-1,
r.." "0
o I
A..N, . õ===--,,µ
µõ,-..., ;$"...... .\ =
1,- ,
ss-..-s...õ...--' = .\\
H Q
0 -...N===== "I's, .....,:::-'
=-....,-..--- = . . = . 5. 00 =õ,.,...õ--
.e ( NNY 11
1 I
o
/1-
-\,..........õ/
, or
H II
,N 0, . ,N ..--J=N. ...-=-=,:, ) \ 0
-, ,===
cr---- = x / 's,,,, ''`..õ..--= N;.====== "*....," ,,,
. t ''.k I!
I I/ \\ h
\:,,,.,......,'N 0
\ _.1
.
[0053] In various embodiments, a compound can be represented by the formula
22
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Ark
(R4)n
0
N/ N
H
H
Formula 1D
wherein R4 can be Rd, SO2Rd, C(=0)Rd, NH C(=0)Rd, Re, ORc, or CF3, wherein RC
can
be H or Ci-Cio hydrocarbyl, Rd can be substituted heterocyclic, unsubstituted
heterocyclic, or unsubstituted carbocyclic, and Re can be substituted
heteroaryl or
substituted phenyl; and n can be 1 or 2. In particular embodiments, R4 can be
CF3,
ORc, or a phenyl group substituted by at least one OCH3 group.
[0054] Additionally, some embodiments of Formula 1D can include compounds
represented by
H
R4 S __,,J / II 0
>_ fait N
H
H N
H
wherein R4 can be: (i) C(=0)Rd and Rd is a pyrrolidonyl group, (ii) SO2Rd and
Rd is a
piperidinyl group, (iii) NHC(=0)Rd and Rd is a phenyl group or a furanyl
group, (iv) an
imidazolyl group, or (v) a thiazole group.
[0055] Further, some embodiments of Formula 1D can include compounds
represented by
23
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411
X H
N ___
I > ___ NEI --j) 11101
H ---N
H
wherein X can be NH or O.
[0056] Embodiments of Formula ID can include compounds represented by
.....,
[ i
.--- -.,,, , ¨ \
,
/
ii
N /5) 0 ,¨=-=-=- \
ii
A :,,õ....- \..., =;\ / \ U
O ¨ =
/2"."\,..--- N."-\==:.\\..õ----S, :\
T
.1 /¨
\
= )---N. /
,;õ ,
0 0 .ii \=
.i.: ..,
.,,,,.. .. , =.....õ ,..",. s =-µ,,.....¨,.. ->-----1
..., - N ........, -....::,,,,...... /
: I. , :,.. ,,.>--- NH \==-1
.\,...---J
,
17 ...................................... k
r,
1 H 0 ¨ I
ii = s?
õ:e \ I
.1\ "
N ...":::`\ A. .."=\ 4: ).-,1
N===e" \NS \w" \ =====''' / \
N.)- = ! = i
µµ..,_ s
b
,j. õ../ ,....:
.õõ .,. .õ, ,, :-:
.,,.:..., sN ,
24
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=s,
t--- = 0 if' .. s; . 0 , \ '.I
.i.i. ; H i. ,
.., :
C: `, Ns:\ ,,,, .. µ 1
..-.1.4'.
1 '
\\., ..............................
0
µN., =N
:::t,
4, .vµ
.-P"' NH
\ ../ i 0 -E. N ..)
\.......,....../
.1
-\": ,":."".. .41",,.. . S - :======4` ,. . /
$
e
e.
ti
::.>.-- tv=-t , ¨ ,,,
1..! ..,-...,, .=
i
1:77'"
h `.\.
11 -,,,,., .44:1 # \\\
\
P .n,-
0 r-----4
-,4
/
,,,,.................õ: I k; .41 '`. , .. .. .... i
1%:---- T
\N =::%--\\,, ...... \,,,, ,..,,, s ">-- S> ' 1
2==', N
,
M
P A ii ,µ V......\
i.> .
µ .),.., 0
(r.----tµ
i
ik,.........if
fiK '....,=e- = = -
\ ''''' ' = µ\\ 6 .
i>-----N/ --V-1...-J--- '=
= N= .
f
F
F ____ =F .
O. /...................N
e
il
,s1.1."-''s,... ....---s-a = )"--- 'C,,k s=---- . N
.\\\:,...... / .N:
-N- ./
= ............................. .k.õ....., - \ - .1.
:
1 li,\
.1 ..
:0 : \. t
\
/.)-------N. \---.1:.
H
,
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(N 0
N * s>_ NH es
N/
H
,
, 0,
H3C0 0 ¨ -CH3
S 0 .0
N
N¨ I
H
, or
S
H3C¨( I 0
S
N
0 N I
H
[0057] In an embodiment, compounds can be represented by the formula
(R4)n
N
0 SNII) 00
H
wherein each R4 can independently be R2, ORa, NR2R3, SR, SORa, SO2Ra, SO2NHRa,
NCORa, C(=0)Ra, CONR2R3, halogen, trihalomethyl, CN, S=0, or nitro and n=1-4.
In
some embodiments, R4 can be optionally substituted heteroaryl. Additionally,
R4 can be
optionally substituted heteroaryl and n can be 1. In various embodiments,
compounds
can be represented by the formula
26
SUBSTITUTE SHEET (RULE 26)
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H- ,C -7 N ii, N o
S>--- .--.---- 0 -----;õ
..õõ...s7
s
[0058] With respect to any relevant structural feature herein, each Ra can
independently be H; optionally substituted hydrocarbyl, such as C1-12 or C1-6
hydrocarbyl; optionally substituted aryl, such as optionally substituted C6-12
aryl,
including optionally substituted phenyl; optionally substituted heteroaryl,
including
optionally substituted C2-12 heteroaryl, such as optionally substituted
pyridinyl, optionally
substituted furyl, optionally substituted thienyl, etc. In some embodiments,
each Ra can
independently be H, or C1-12 alkyl, including: linear or branched alkyl having
the formula
CaHa.i, or cycloalkyl having the formula CaHa-1, wherein a is 1, 2, 3, 4, 5,
6, 7, 8, 9, 10,
11, or 12, such as linear or branched alkyl of the formula: CH3, C2H5, C3H7,
C4H9, C5H11,
C61-113, C7H15, C8H17, C9H19, C10H21, etc., or cycioalkyl of the formula:
C3H5, C4H7, C5H9,
C6H11, C7H13, C8H15, C9I-117, C10H19, etc.
[0059] With respect to Ra, in some embodiments, the aryl group can be
substituted
with halogen, trihalomethyl, alkoxy, alkylamino, OH, CN, alkylthio, arylthio,
sulfoxide,
arylsulfonyl, alkylsulfonyl, carboxylic acid, nitro or acylamino.
[0060] With respect to Ra, in some embodiments, the heteroaryl group can be
single or
fused. In some embodiments, the single heteroaryl group can be imidazole. In
some
embodiments, the fused heteroaryl group can be benzimidazole. In some
embodiments,
the heteroaryl group can be substituted with halogen, trihalomethyl, alkoxy,
alkylamino,
OH, CN, alkylthio, arylthio, sulfoxide, arylsulfonyl, alkylsulfonyl,
carboxylic acid, nitro or
acylamino. In some embodiments, the alkyl group can be branched, cyclic or
polycyclic.
[0061] With respect to Ra, a hydrocarbyl can be alkyl, alkenyl, or alkynyl. In
some
embodiments, the alkyl group can be substituted with halogen, trihalomethyl,
alkoxy,
alkylamino, OH, CN, heteroaryl, alkylthio, arylthio, sulfoxide, arylsulfonyl,
alkylsulfonyl,
carboxylic acid, nitro, or acylamino. In some embodiments, the heteroaryl
group can be
single or fused. In some embodiments, the single heteroaryl group can be
imidazole. In
27
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some embodiments, the fused heteroaryl group can be benzimidazole. In some
embodiments, the alkenyl group can be branched, cyclic or polycyclic. In some
embodiments, the alkenyl group can be substituted with halogen, trihalomethyl,
alkoxy,
alkylamino, OH, CN, heteroaryl, alkylthio, arylthio, sulfoxide, arylsulfonyl,
alkylsulfonyl,
carboxylic acid, nitro, or acylamino.
[0062] With respect to any relevant structural feature herein, Rb can be H, or
C1-3
hydrocarbyl, such as CH3, C2H5, C3H7, cyclopropyl, CH=CH2, CH2CH=CH2,
CH2CCH, etc.
[0063] With respect to any relevant structural feature herein, RC can be H, or
C1-3 alkyl,
such as CH3, C2H5, C3H7, cyclopropyl, etc. In some embodiments, RC can be H.
[0064] With respect to any relevant formula or structural depiction herein,
such as
Formula 1, Formula 2, Formula 3, or Formula 4, R1 can be Ra, 0R2 or NR2R3. In
some
embodiments, R1 can be optionally substituted phenyl. In some embodiments, R1
can
be unsubstituted phenyl. In some embodiments, R1 can be optionally substituted
naphthyl. In some embodiments, R1 can be unsubstituted naphthyl.
In other
embodiments, R1 can be
R7
R8
\R9
-(1 Rio
[0065] In some embodiments, R1 can be.
,R1 2
R7
1 0
R1
28
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[0066] In some embodiments, R1 can be
R,7 R8
. >,--c
---c\ 9
11
R R1 o
[0067] In some embodiments, R1 can be
\
t
/1 / R12
1
' 13
[0068] In some embodiments, R1 can be
R.15
R16
i
R7 . ......::......._
11
R
18
R
Ri'l \ io
R =
[0069] In some embodiments, Ri can be
29
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R15
R16
R7
18
[0070] In some embodiments, R1 can be
Ri9
R20
,R22
[0071] With respect to any relevant structural feature herein, R2 can be Ra,
CORa, or
SO2Ra. In some embodiments, R2 can be H, methyl, ethyl, a propyl (e.g. n-
propyl,
isopropyl, etc.), cyclopropyl, a butyl, cyclobutyl or an isomer thereof, a
pentyl,
cyclopentyl or an isomer thereof, a hexyl, a cyclohexyl or an isomer thereof,
etc. In
some embodiments, R2 can be H.
[0072] With respect to any relevant structural feature herein, R3 can be Ra,
C(=0)Ra, or
SO2Ra. In some embodiments, R3 can be H, methyl, ethyl, a propyl (e.g. n-
propyl,
isopropyl, etc.), cyclopropyl, a butyl, cyclobutyl or an isomer thereof, a
pentyl,
cyclopentyl or an isomer thereof, a hexyl, a cyclohexyl or an isomer thereof,
etc. In
some embodiments, R3 can be H.
[0073] With respect to any relevant structural feature herein, each R4 can
independently be R2, ORa, C(=0)Ra, CO2Ra, OCORa, CONR2R3, NR2R3, NRbC(=0)Ra,
SRa, SORa, SO2Ra, SO2NHRa, SO2NRaRb, NC(=0)Ra, halogen, trihalomethyl, CN,
S=0,
nitro, or C2-5 heteroaryl. In some embodiments, R4 can be H.
[0074] Generally R5 and R7-R22, can be H or any substituent, such as a
substituent
having from 0 to 6 carbon atoms and from 0 to 5 heteroatoms independently
selected
from: 0, N, S, F, Cl, Br, and I, and/or having a molecular weight of 15 g/mol
to 300
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g/mol. Any of R5 and R7-R22 can include: a) 1 or more alkyl moieties
optionally
substituted with, or optionally connected by, b) 1 or more functional groups,
such as
C=C, C---=C, CO, CO2, CON, NCO2, OH, SH, 0, S, N, N=C, F, Cl, Br, I, CN, NO2,
CO2H,
NH2, etc.; or can be a substituent having no alkyl portion, such as F, Cl, Br,
I, NO2, CN,
NH2, OH, COH, CO2H, etc.
[0075] With respect to any relevant structural feature herein, R5 can be Ra,
CORa,
SO2Ra, or may not be present. Some examples of R5 can include H or C1-3 alkyl,
such
as CH3, C2H5, C7, cyclopropyl, etc. In some embodiments, R5 can be CH3. In
some
embodiments, R5 is H.
[0076] With respect to any relevant formula or structural depiction above,
some
examples of R19 can include Rb, ORb, SRb, C(=0)Rb, CO2Rb, OC(=0)Rb, NRbW,
C(=0)NRbRc, NRbC(=0)Rc, SO2NRbRc, CF3, CN, NO2, F, Cl, Br, I, or C2-5
heterocyclyl.
In some embodiments, R19 can be H, CH3, CH2CH3, Cl, Br, OH, OCH3, SCH3, NH2,
NHCH3, N(CH3)2, SO2NH2, morpholino, CH2C-iECH, or NO2. In some embodiments,
R19
can be H.
-N 0
morpholino
[0077] With respect to any relevant formula or structural depiction above,
some
examples of R5 can include Rb, or as depicted below, C(=0)Rb, CO2Rb,
C(=0)NRbRc,
NRbC(=0)Rc, SO2NRbRc, CF3, CN, or C2-5 heterocyclyl. In some embodiments, R5
can
be H, CH3, CH2CH3, SO2NH2, or CH2C---CH. In some embodiments, R5 can be H,
CH3,
CH2CH3, CH2CH2CH3, CH2CH=CH2, or CH2C=CH. In some embodiments, R5 is
CH2C---- CH. In some embodiments, R5 can be H.
0 Rb
0 0 Rc
Rc
Rb N
.R-
VjlµN\ Rb 0* ".
Rb 0
CORb CO2Rb CONRbRc NRbCORc
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0 0
Rc
Rb
SO2NRbRc
[0078] With respect to any relevant formula or structural depiction above,
some
examples of R7 can include Rb, ORb, SRb, C(=0)Rb, CO2Rb, OC(=0)Rb, NRbRc,
C(=0)NRbRc, NRbC(=0)Rc, SO2NRbRc, CF3, CN, NO2, F, Cl, Br, I, or C2-5
heterocyclyl,
In some embodiments, R7 can be H, CH3, CH2CH3, CI, Br, OH, OCH3, SCH3, NH2,
NHCH3, N(CH3)2, SO2NH2, morpholino, CH2C------ CH, or NO2. In some
embodiments, R7
can be H.
[0079] With respect to any relevant formula or structural depiction above,
some
examples of R8 can include Rb, ORb, SRb, C(=0)Rb, CO2Rb, OC(=0)Rb, NRbW,
C(=0)NRbRe, NRbC(=0)Re, SO2NRbRc, CF3, CN, NO2, F, Cl, Br, I, or C2-5
heterocyclyl.
In some embodiments, R8 can be H, CH3, CH2CH3, Cl, Br, OH, OCH3, SCH3, NH2,
NHCH3, N(CH3)2, SO2NH2, morpholino, CH2CE-:CH, or NO2. In some embodiments, R8
can be H, Cl or Br. In some embodiments, R8 can be Cl. In some embodiments, R8
can
be Br. In some embodiments, R8 can be H.
[0080] With respect to any relevant formula or structural depiction above,
some
examples of R9 can include Rb, ORb, SRb, C(=0)Rb, CO2Rb, OC(=0)Rb, NRbRc,
C(=0)NRbRe, NRbC(=0)Rc, SO2NRbRc, CF3, CN, NO2, F, Cl, Br, I, or C2-5
heterocyclyl.
In some embodiments, R9 can be H, CH3, CH2CH3, Cl, Br, OH, OCH3, SCH3, NH2,
NHCH3, N(CH3)2, SO2NH2, morpholino, CH2CECH, or NO2. In some embodiments, R9
can be H, Cl, or SO2NH2. In some embodiments, R9 can be H. In some
embodiments,
R9 can be Cl. In some embodiments, R9 can be SO2NH2. In some embodiments, R9
can
be H.
[0081] In some embodiments, R8 and R9 can be joined together to form: IL017
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[0082] 1Nith respect to any relevant formula or structural depiction above,
some
examples of R1 can include Rb, ORb, SRb, C(=0)Rb, CO2Rb, OC(=0)Rb, NRbRc,
C(=0)NRbRc, NRbC(.0)Rc, SO2NRbRc, CF3, CN, NO2, F, Cl, Br, I, or 02-5
heterocyclyl.
In some embodiments, R19 can be H, CH3, CH2CH3, Cl, Br, OH, OCH3, SCH3, NH2,
NHCH3, N(CH3)2, SO2NH2, morpholino, CH2C 7=CH, or NO2. In some embodiments, R1
can be H or Cl. In some embodiments, R19 can be H. In some embodiments, R1
can be
Cl. In some embodiments, R8 and R1 can be Cl.
[0083] With respect to any relevant formula or structural depiction above,
some
examples of R11 can include Rb, ORb, SRb, C(=0)Rb, CO2Rb, OC(=0)Rb, NRbRc,
C(=0)NRbRc, NRbC(=0)Rc, CF3, CN, NO2, F, Cl, Br, or I. In some embodiments,
R11
can be H, CH3, CH2CH3, Cl, Br, OH, OCH3, SCH3, NH2, NHCH3, N(CH3)2, CH2C7==CH,
or
NO2. In some embodiments, R11 can be H.
[0084] In some embodiments, R7 and R11 can be H. In some embodiments, R7, R9,
and
R11 can be H. In some embodiments, R7, R10, and R11 can be H. In some
embodiments,
R7, R8, R19, and R11 can be H. In some embodiments, R7, R8, R9, and R11 can be
H. In
some embodiments, R7, R8, R9, R10, and R11 can be H.
[0085] With respect to any relevant formula or structural depiction above,
some
examples of R15 can include Rb, ORb, SRb, C(=0)Rb, CO2Rb, OC(=0)Rb, NRbW,
C(=0)NRbRc, NRbC(=0)Rc, SO2NRbRc, CF3, CN, NO2, F, CI, Br, or C2-5
heterocyclyl. In
some embodiments, R15 can be H, CH3, CH2CH3, Cl, Br, OH, OCH3, SCH3, NH2,
NHCH3, N(CH3)2, SO2NH2, morpholino, CH2C=CH, or NO2. In some embodiments, R15
can be H.
[0086] With respect to any relevant formula or structural depiction above,
some
examples of R16 can include Rb, ORb, SRb, C(-7-0)Rb, CO2Rb, OC(=O)Rb, NRbRc,
C(=0)NRbRc, NRbC(=0)Rc, SO2NRbRc, CF3, CN, NO2, F, CI, Br, I, or C2-5
heterocyclyl.
In some embodiments, R16 can be H, CH3, CH2CH3, Cl, Br, OH, OCH3, SCH3, NH2,
NHCH3, N(CH3)2, SO2NH2, CH2C,7-7 CH, or NO2. In some embodiments, R16 can be
H.
[0087] With respect to any relevant formula or structural depiction above,
some
examples of R17 can include Rb, ORb, SRb, C(=0)Rb, CO2Rb, OC(7---0)Rb, NRbRb,
C(=0)NRbRc, NRbC(=0)Rc, SO2NRbRc, CF3, CN, NO2, F, CI, Br, I, or C2-5
heterocyclyl.
In some embodiments, R17 can be H, CH3, Cl, Br, OH, OCH3, SCH3, NH2, NHCH3,
33
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N(CH3)2, SO2NH2, morpholino, CH2C-7-= CH, or NO2. In some embodiments, R17 can
be
H.
[0088] With respect to any relevant formula or structural depiction above,
some
examples of R18 can include Rb, ORb, SRb, C(=0)Rb, CO2Rb, OC(=0)Rb, NRbRc,
C(=0)NRbRc, NRbC(=0)Rc, SO2NRbRb, CF3, CN, NO2, F, CI, Br, I, or C2-5
heterocyclyl.
In some embodiments, R18 can be H, CH3, CH2CH3, Cl, Br, OH, OCH3, SCH3, NH2,
NHCH3, N(CH3)2, SO2NH2, morpholino, or NO2. In some embodiments, R18 can be H.
[0089] In some embodiments, R18, R15, R17, and R18 can be H.
[0090] With respect to any relevant formula or structural depiction above,
some
examples of R12 can include Rb, ORb, SRb, C(=0)Rb, CO2Rb, OC(=0)Rb, NRbRc,
C(=0)NRbRc, NRbC(=0)Rc, SO2NRbRe, CF3, CN, NO2, F, Cl, Br, I, or C2_5
heterocyclyl.
In some embodiments, R12 can be H, CH3, CH2CH3, CI, Br, OH, OCH3, SCH3, NH2,
NHCH3, N(CH3)2, SO2NH2, CH2C----=CH, or NO2. In some embodiments, R12 can be H
or
SO2NH2. In some embodiments, R12 can be H. In some embodiments, R12 can be
SO2NH2.
[0091] With respect to any relevant formula or structural depiction above,
some
examples of R13 can include Rb, ORb, SRb, C(=0)Rb, CO2Rb, OC(=0)Rb, NRbRc,
C(=0)NRbRc, NRbC(=0)Rc, SO2NRbRc, CF3, CN, NO2, F, Cl, Br, I, or C2-5
heterocyclyl.
In some embodiments, R13 can be H, CH3, CH2CH3, Cl, Br, OH, OCH3, SCH3, NH2,
NHCH3, N(CH3)2, SO2NH2, morpholino, or NO2. In some embodiments, R13 can be H.
[0092] In some embodiments, R12 and R13 can be H.
[0093] With respect to any relevant formula or structural depiction above,
some
examples of R2 can include Rb, ORb, SRb, C(=0)Rb, CO2Rb, OC(=0)Rb, NRbRc,
C(=0)NRbRc, NR C(=0)Rc, SO2NRbRc, CF3, CN, NO2, F, Cl, Br, I, or C2-8
heterocyclyl.
In some embodiments, R2 can be H, CH3, CH2CH3, Cl, Br, OH, OCH3, SCH3, NH2,
NHCH3, N(CH3)2, SO2NH2, morpholino, CH2C---CH, or NO2. In some embodiments, R2
can be H, CH2CH3, OCH3, N(CH3)2, morpholino, or SCH3. In some embodiments, R2
can be H. In some embodiments, R2 can be CH2CH3. In some embodiments, R2 can
be OCH3. In some embodiments, R2 can be CN(CH3)2. In some embodiments, R2
can
be morpholino. In some embodiments, R2 can be SCH3.
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[0094] With respect to any relevant formula or structural depiction above,
some
examples of R22 can include Rb, ORb, SRb, CF3, CN, NO2, F, Cl, Br, I, or C2-5
heterocyclyl. In some embodiments, R22 can be H, CH3, or CH2CH3. In some
embodiments, R22 can be H.
[0095] In some embodiments, R19 and R22 can be H.
[0096] With respect to any relevant formula or structural depiction above,
some
examples of R14 can include Rb, ORb, SRb, C(=0)Rb, CO2Rb, OC(=0)Rb, NRbRc,
C(=0)NRbRc, NRbC(=0)Re, SO2NRbRc, CF3, CN, NO2, F, Cl, Br, or I. In some
embodiments, R39 can be H, CH3, CH2CH3, Cl, Br, OH, OCH3, SCH3, NH2, NHCH3,
N(CH3)2, SO2NH2, CH2C-----CH, or NO2. In some embodiments, R14 can be H.
[0097] With respect to any relevant formula or structural depiction above,
examples of
R13 can include Rb, ORb, SRb, C(=0)Rb, CO2Rb, OC(=0)Rb, NRbRc, C(=0)NRbRc,
NRbC(=0)Rc, SO2NRbRc, CF3, CN, NO2, F, Cl, Br, I, or C2-5 heterocyclyl. In
some
embodiments, R13 can be H, CH3, CH2CH3, Cl, Br, OH, OCH3, SCH3, NH2, NHCH3,
N(CH3)2, SO2NH2, morpholino, CH2C=CH, or NO2. In some embodiments, R13 can be
H.
[0098] With respect to any relevant formula or structural depiction above,
some
examples of R12 can include Rb, ORb, SRb, C(=0)Rb, CO2Rb, OC(=0)Rb, NRbRc,
C(=0)NRbRc, NRbC(=0)Rc, SO2NRbRc, CF3, CN, NO2, F, Cl, Br, I, or C2-5
heterocyclyl.
In some embodiments, R12 can be H, CH3, CH2CH3, Cl, Br, OH, OCH3, SCH3, NH2,
NHCH3, N(CH3)2, SO2NH2, morpholino, CH2C=-=CH, or NO2. In some embodiments,
R12
can be H or NO2. In some embodiments, R12 can be H. In some embodiments, R12
can
be NO2.
[0099] In some embodiments, R14, R13, and R12 can be H. In some embodiments,
R13
and R12 can be H.
[0100] As suggested by the list of compounds in Table 1, in some instances,
the
substituent that is joined to an aromatic carbon atom is H.
[0101] Specific embodiments of the compounds disclosed herein have the
structures
shown in Table 1.
Table 1
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Structure Compound ID
H3c 1
21------- N
S 100 s >¨T 0 --) =
N
H ___________________________________
Br
p-- N 2
s o
s \
el il le.
N
H
H
H3C -......N o)./ NI .....1
0 Br 3
S N 0
p"---- N 4
s o
0\
Ti
el ?¨
N
H
N S I 0
N ....1
6
H
NI
0
or N ---,1
0 0 Br
S
7
N, il 0
0 opi s \ - rj le
8
H
1
H3C N --__ = ).../ N s....1 11110 Cl
S N 0
36
SUBSTITUTE SHEET (RULE 26)
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PCT/US2015/030014
CH3 9
r---io
o
N`..,...,....1=CH3
H 0 \µ0
N 0 1
H3C ..-_. = r N ----,10
S
H
1 00 0.,,....,..-,õõNLNC.0 10
(S 0N 0
11
0
0 P
0 0 I
N
H
H 12
10010 s>- ri
o ap
0
N
H
13
( o)T 0 s )_ ) Ia.
/ T _____________________________________
N
H
4
110 4 NH
0
S>_ irj
IIP
N
0 N
H
0 o
O
S
N
../j
101
H
37
SUBSTITUTE SHEET (RULE 26)
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F 16
F F
0
S
AP
H _____________________________________
17
H3c 44 1
H3c o 10 j
S\ .
cH3 N>¨ i
H
N 18
0
\ N 0 s>_ N=
H
,0 0 0, 19
H3c-- --cH3
o
s
0 N>¨ 7 el
H
H3cõ ...1 20
0
S N\/CH3
H
1
H3CN . iii...õ. N ....10
\
S21
H3c-4, 1
S
N ) 101
H
[0102] Unless stereochemistry is unambiguously depicted, any structure,
formula, or
name for a compound can refer to any stereoisomer or any mixture of
stereoisomers of
the compound.
38
SUBSTITUTE SHEET (RULE 26)
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[0103] Unless otherwise indicated, any reference to a compound herein by
structure,
formula, name or any other means, includes pharmaceutically acceptable salts,
such as
sodium, potassium, and ammonium salts; prodrugs, such as ester prodrugs;
alternate
solid forms, such as polymorphs, solvates, hydrates, etc.; tautomers; isomers;
or, any
other chemical species that may rapidly convert to a compound described herein
under
conditions in which the compounds are used as described herein.
[0104] As used herein, the term "pharmaceutically acceptable salt" refers to
pharmaceutical salts that are, within the scope of sound medical judgment,
suitable for
use in contact with the tissues of subjects without undue toxicity,
irritation, and allergic
response, and are commensurate with a reasonable benefit/risk ratio.
Pharmaceutically
acceptable salts are well known in the art. In one embodiment, the
pharmaceutically
acceptable salt is a sulfate salt. For example, S. M. Berge, et al. describes
pharmaceutically acceptable salts in J. Pharm. Sci., 1977, 66:1-19.
[0105] Suitable pharmaceutically acceptable acid addition salts can be
prepared from
an inorganic acid or an organic acid. Examples of such inorganic acids are
hydrochloric,
hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid.
Appropriate
organic acids can be selected from aliphatic, cycloaliphatic, aromatic,
arylaliphatic,
heterocyclic, carboxylic and sulfonic classes of organic acids, examples of
which are
formic, acetic, propionic, succinic, glycolic, gluconic, maleic, embonic
(pamoic),
methanesulfonic, ethanesulfonic, 2-hyd
roxyethanesulfonic, pantothenic,
benzenesulfonic, toluenesulfonic, sulfanilic, mesylic,
cyclohexylaminosulfonic, stearic,
algenic, p-hydroxybutyric, malonic, galactic, and galacturonic acid.
Pharmaceutically
acceptable acidic/anionic salts also include, the acetate, benzenesulfonate,
benzoate,
bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate,
chloride,
citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate,
glyceptate,
gluconate, glutamate, glycollylarsanilate, hexylresorcinate,
hydrobromide,
hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate,
malate,
maleate, malonate, mandelate, mesylate, methylsulfate, mucate, napsylate,
nitrate,
pamoate, pantothenate, phosphate/diphospate, polygalacturonate, salicylate,
stearate,
subacetate, succinate, sulfate, hydrogensulfate, tannate, tartrate, teoclate,
tosylate, and
triethiodide salts.
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[0106] Suitable pharmaceutically acceptable base addition salts include
metallic salts
made from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc
or
organic salts made from N,N'-dibenzylethylene-diamine, chloroprocaine,
choline,
diethanolamine, ethylenediamine, N-methylglucamine, lysine, arginine, and
procaine. All
of these salts can be prepared by conventional means from the corresponding
compound represented by the disclosed compounds by treating, for example, the
disclosed compounds with the appropriate acid or base. Pharmaceutically
acceptable
basic/cationic salts also include, the diethanolamine, ammonium, ethanolamine,
piperazine, and triethanolamine salts.
[0107] A pharmaceutically acceptable salt includes any salt that retains the
activity of
the parent compound and is acceptable for pharmaceutical use. A
pharmaceutically
acceptable salt also refers to any salt which may form in vivo as a result of
administration of an acid, another salt, or a prodrug which is converted into
an acid or
salt.
[0108] A prodrug includes a compound which is converted to a therapeutically
active
compound after administration, such as by hydrolysis of an ester group or some
other
biologically labile group.
[0109] "Functional group" refers to an atom or a group of atoms that have
similar
chemical properties whenever they occur in different compounds, and as such
the
functional group defines the characteristic physical and chemical properties
of families
of organic compounds.
[0110] Unless otherwise indicated, when any compound or chemical structural
feature
(collectively referred to herein as a "compound"), such as for example alkyl,
aryl, etc., is
referred to as being "optionally substituted," that compound can have no
substituents (in
which case it is "unsubstituted"), or it can include one or more substituents
(in which
case it is "substituted"). The term "substituent" has the ordinary meaning
known to one
of ordinary skill in the art. In some embodiments, the substituent can be an
ordinary
organic moiety known in the art, which can have a molecular weight (e.g., the
sum of
the atomic masses of the atoms of the substituent) of 15 g/mol to 50 g/mol, 15
g/mol to
100 g/mol, 15 g/mol to 150 g/mo1,15 g/mol to 200 g/mol, 15 g/mol to 300 g/mol,
or 15
g/mol to 500 g/mol. In some embodiments, the substituent includes: 0-30, 0-20,
0-10, or
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0-5 carbon (C) atoms; and/or 0-30, 0-20, 0-10, or 0-5 heteroatoms including N,
0, S, Si,
F, CI, Br, or I; provided that the substituent includes at least one atom
including C, N, 0,
S, Si, F, CI, Br, or I in a substituted compound. Examples of substituents can
include
alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl,
heteroaryl,
hydroxy, alkoxy, aryloxy, acyl, acyloxy, alkylcarboxylate, thiol, alkylthio,
cyano, halo,
thiocarbonyl, 0-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,
N-amido, S-sulfonamido, N-sulfonamido, isocyanato, thiocyanato,
isothiocyanato, nitro,
silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxyl,
trihalomethanesulfonyl,
trihalomethanesulfonamido, amino, etc. For convenience, the term "molecular
weight" is
used with respect to a moiety or part of a molecule to indicate the sum of the
atomic
masses of the atoms in the moiety or part of a molecule, even though it may
not be a
complete molecule.
[0111] "Hydrocarbyl" has the broadest meaning generally understood in the art,
and
can include a moiety composed of carbon and hydrogen. Some examples can
include
alkyl, alkenyl, alkynyl, aryl, etc., and combinations thereof, and can be
linear, branched,
cyclic, or a combination thereof. Hydrocarbyl can be bonded to any other
number of
moieties (for example, can be bonded to one other group, such as ¨CH3,
¨CH=CH2,
etc.; two other groups, such as ¨phenyl-, -CaC-, etc.; or any number of other
groups)
that the structure can bear, and in some embodiments, can contain from one to
thirty-
five carbon atoms. Examples of hydrocarbyl groups include C1 alkyl, C2 alkyl,
C2
alkenyl, C2 alkynyl, C3 alkyl, C3 alkenyl, C3 alkynyl, C4 alkyl, C4 alkenyl,
C4 alkynyl,
C5 alkyl, C5 alkenyl, C5 alkynyl, C6 alkyl, C6 alkenyl, C6 alkynyl, phenyl,
etc.
[0112] "Alkyl" has the broadest meaning generally understood in the art, and
can
include a moiety composed of carbon and hydrogen containing no double or
triple
bonds and not having any cyclic structure. Alkyl can be linear alkyl, branched
alkyl,
cycloalkyl, or a combination thereof, and in some embodiments, can contain
from one to
thirty-five carbon atoms. In some embodiments, alkyl can include C1-10 linear
alkyl, such
as methyl (-CH3), ethyl (-CH2CH3), n-propyl (-CH2CH2CH3), n-butyl (-
CH2CH2CH2CH3),
n-pentyl (-CH2CH2CH2CH2CH3), n-hexyl (-CH2CH2CH2CH2CH2CH3), etc.; C3-1O
branched alkyl, such as C3H7 (e.g. iso-propyl), C41-19 (e.g., branched butyl
isomers),
C5H1 (e.g., branched pentyl isomers), C6H13 (e.g., branched hexyl isomers),
C7H15
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(e.g., branched heptyl isomers), etc.; C3-10 cycloalkyl, such as C3H5 (e.g.
cyclopropyl),
C4H7 (e.g., cyclobutyl isomers such as cyclobutyl, methylcyclopropyl, etc.),
C5H9 (e.g.,
cyclopentyl isomers such as cyclopentyl, methylcyclobutyl,
dimethylcyclopropyl, etc.)
C61-111 cyclohexyl isomers), C7H13 (e.g., cycloheptyl isomers), etc.; C3-
12
bicycloalkyl such as decahydronaphthyl, and norbornyl; and the like.
[0113] "Alkyl," "alkenyl" and "alkynyl" refer to substituted and unsubstituted
alkyls,
alkenyls and alkynyls, respectively. An alkyl group can be optionally
substituted as
defined herein.
[0114] Substituted alkyls, alkenyls, and alkynyls refer to alkyls, alkenyls,
and alkynyls
substituted with one to five substituents including H, alkyl, aryl, alkenyl,
alkynyl,
arylalkyl, alkoxy, aryloxy, arylalkoxy, alkoxyalkylaryl, alkylamino,
arylamino, NH2, OH,
CN, NO2, OCF3, CF3, F, 1-amidine, 2-amidine, alkylcarbonyl, morpholinyl,
piperidinyl,
dioxanyl, pyranyl, heteroaryl, furanyl, thiophenyl, tetrazolo, thiazolyl,
isothiazolyl,
imidazolyl, thiadiazolyl, thiadiazole S-oxide, thiadiazole S,S-dioxide,
pyrazolo, oxazolyl,
isoxazolyl, pyridinyl, pyrimidinyl, quinolinyl, isoquinolinyl, SR, SOR, SO2R,
CO2R, COR,
CONITR", CSNR'R", and SOnNR'R". As used herein, R, R', and R" can include R
groups described in this disclosure, such as Ra, Rb, Rc, Rd, Re, R2, or R3.
[0115] Either alone or in combination, "alkynyl" refers to a functional group
including a
straight-chain or branched-chain hydrocarbon containing from 2 to 20 carbon
atoms and
having one or more carbon-carbon triple bonds and not having any cyclic
structure. An
alkynyl group may be optionally substituted as defined herein. Examples of
alkynyl
groups include ethynyl, propynyl, hydroxypropynyl, butynyl, butyn-1-yl, butyn-
2-yl, 3-
methylbutyn-1-yl, pentynyl, pentyn-1-y!, hexynyl, hexyn-2-y!, heptynyl,
octynyl, nonynyl,
decynyl, undecynyl, dodecynyl, tridecynyl, tetradecynyl, pentadecynyl,
hexadecynyl,
heptadecynyl, octadecynyl, nonadecynyl, eicosynyl, and the like.
[0116] "Alkylene", alone or in combination, refers to a saturated aliphatic
group derived
from a straight or branched chain saturated hydrocarbon attached at two or
more
positions, such as methylene (¨CH2¨). Unless otherwise specified, "alkyl" can
include
"alkylene" groups.
[0117] Either alone or in combination "alkylcarbonyl" or "alkanoyl" refer to a
functional
group including an alkyl group attached to the parent molecular moiety through
a
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carbonyl group. Examples of alkylcarbonyl groups can include methylcarbonyl,
ethylcarbonyl, and the like.
[0118] Either alone or in combination, "heteroalkyl" refers to a functional
group
including a straight-chain, branched-chain, or cyclic hydrocarbon containing
from 1 to 20
atoms linked exclusively by single bonds, where at least one atom in the chain
is a
carbon and at least one atom in the chain is 0, S, N, or any combination
thereof. The
heteroalkyl group can be fully saturated or contain from 1 to 3 degrees of
unsaturation.
The non-carbon atoms can be at any interior position of the heteroalkyl group,
and up to
two non-carbon atoms can be consecutive, such as, e.g., ¨CH2¨NH¨OCH3. In
addition,
the non-carbon atoms can optionally be oxidized and the nitrogen can
optionally be
quaternized. Examples of heteroalkyl groups can include morpholine,
azanorbornane,
tetrahydrofuran, and the like.
[0119] Either alone or in combination, "alkyloxy" or "alkoxy" refer to a
functional group
including an alkyl ether group. Examples of alkoxys can include methoxy,
ethoxy, n-
propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, and the
like.
[0120] Either alone or in combination, "hydroxy" refers to the functional
group hydroxyl
(¨OH).
[0121] Either alone or in combination, "carboxyl" or "carboxy" refers to the
functional
group ¨C(=0)0H or the corresponding "carboxylate" anion ¨C(=0)0-. Examples
include formic acid, acetic acid, oxalic acid, and benzoic acid. An "0-
carboxyl" group
refers to a carboxyl group having the general formula RCOO, wherein R is an
organic
moiety or group. A "C-carboxyl" group refers to a carboxyl group having the
general
formula COOR, wherein R is an organic moiety or group.
[0122] Either alone or in combination, "oxo" refers to the functional group
=O.
[0123] "Ca rbocycl ic" has the broadest meaning generally understood in the
art, and
includes a ring or ring system wherein the ring atoms are all carbon. Examples
can
include phenyl, naphthyl, anthracenyl, cycloalkyl, cycloalkenyl, cycloalkynyl,
etc., and
combinations thereof.
[0124] "Heterocyclic" has the broadest meaning generally understood in the
art, and
includes a ring or ring system wherein at least one of the ring atoms is not
carbon, such
as N, 0, S, etc. Examples can include heteroaryl, cycloheteroalkyl,
cycloheteroalkenyl,
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cycloheteroalkynyl, cyclic heteroalkyl, etc., and combinations thereof.
Examples of
heterocyclic systems can include quinoline, tetrahydroisoquinoline,
tetrahydropyran,
imidazole, thiophene, dihydrobenzofuran, and the like.
[0125] Either alone or in combination, "cycloalkyl," "carbocyclicalkyl" and
"carbocyclealkyr refer to a functional group including a substituted or
unsubstituted non-
aromatic hydrocarbon with a non-conjugated cyclic molecular ring structure of
3 to 12
carbon atoms linked exclusively with carbon-carbon single bonds in the carbon
ring
structure. A cycloalkyl group can be monocyclic, bicyclic or polycyclic, and
can
optionally include one to three additional ring structures, such as, e.g., an
aryl, a
heteroaryl, a cycloalkenyl, a heterocycloalkyl, or a heterocycloalkenyl.
[0126] Either alone or in combination, "lower cycloalkyl" refers to a
functional group
including a monocyclic substituted or unsubstituted non-aromatic hydrocarbon
with a
non-conjugated cyclic molecular ring structure of 3 to 6 carbon atoms linked
exclusively
with carbon-carbon single bonds in the carbon ring structure. Examples of
lower
cycloalkyl groups can include cyclopropyl, cyclobutyl, cyclopentyl, and
cyclohexyl.
[0127] "Aryl" has the broadest meaning generally understood in the art, and
can
include an aromatic ring or aromatic ring system. An aryl group can be
monocyclic,
bicyclic or polycyclic, and may optionally include one to three additional
ring structures;
such as, for example, a cycloalkyl, a cycloalkenyl, a heterocycloalkyl, a
heterocycloalkenyl, or a heteroaryl. The term "aryl" includes phenyl
(benzenyl),
thiophenyl, indolyl, naphthyl, tolyl, xylyl, anthracenyl, phenanthryl,
azulenyl, biphenyl,
naphthalenyl, 1-methylnaphthalenyl, acenaphthenyl, acenaphthylenyl,
anthracenyl,
fluorenyl, phenalenyl, phenanthrenyl, benzo[a]anthracenyl,
benzo[c]phenanthrenyl,
chrysenyl, fluoranthenyl, pyrenyl, tetracenyl (naphthacenyl), triphenylenyl,
anthanthrenyl, benzopyrenyl, benzo[a]pyrenyl,
benzo[e]fluoranthenyl,
benzo[ghi]perylenyl, benzo[j]fluoranthenyl, benzo[k]fluoranthenyl,
corannulenyl,
coronenyl, dicoronylenyl, helicenyl, heptacenyl, hexacenyl, ovalenyl,
pentacenyl,
picenyl, perylenyl, tetraphenylenyl, etc.
[0128] Additionally, either alone or in combination, "aryl," "hydrocarbyl
aryl" or "aryl
hydrocarbon" can refer to a functional group including a substituted or
unsubstituted
aromatic hydrocarbon with a conjugated cyclic molecular ring structure of 3 to
12 carbon
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atoms. Substituted aryl refers to aryls substituted with one to five
substituents including
H, lower alkyl, aryl, alkenyl, alkynyl, arylalkyl, alkoxy, aryloxy,
arylalkoxy, alkoxyalkylaryl,
alkylamino, arylamino, NH2, OH, CN, NO2, OCF3, CF3, Br, Cl, F, 1-amidino, 2-
amidino,
alkylcarbonyl, morpholino, piperidinyl, dioxanyl, pyranyl, heteroaryl,
furanyl, thiophenyl,
tetrazolo, thiazole, isothiazolo, imidazolo, thiadiazole, thiadiazole S-oxide,
thiadiazole
S,S-dioxide, pyrazolo, oxazole, isoxazole, pyridinyl, pyrimidinyl, quinoline,
isoquinoline,
SR, SOR, SO2R, CO2R, COR, CONR'R", CSNRJR", SOnNR'R", etc.
[0129] Either alone or in combination, "lower aryl" refers to a functional
group including
a substituted or unsubstituted aromatic hydrocarbon with a conjugated cyclic
molecular
ring structure of 3 to 10 carbon atoms. Examples of lower aryl groups can
include
phenyl and naphthyl.
[0130] Either alone or in combination, "heteroaryl" refers to a functional
group including
a substituted or unsubstituted aromatic hydrocarbon with a conjugated cyclic
molecular
ring structure of 3 to 12 atoms, where at least one atom in the ring structure
is a carbon
and at least one atom in the ring structure is 0, S, N, or any combination
thereof. A
heteroaryl group can be monocyclic, bicyclic or polycyclic, and may optionally
include
one to three additional ring structures, such as, e.g., an aryl, a cycloalkyl,
a
cycloalkenyl, a heterocycloalkyl, or a heterocycloalkenyl. Examples of
heteroaryl groups
can include acridinyl, benzidolyl, benzimidazolyl, benzisoxazolyl,
benzodioxinyl,
dihydrobenzodioxinyl, benzodioxolyl, 1,3-benzodioxolyl, benzofuryl,
benzoisoxazolyl,
benzopyranyl, benzothiophenyl, benzo[c]thiophenyl, benzotriazolyl,
benzoxadiazolyl,
benzoxazolyl, benzothiadiazolyl, benzothiazolyl, benzothienyl, carbazolyl,
chromonyl,
cinnolinyl, dihydrocinnolinyl, coumarinyl, dibenzofuranyl, furopyridinyl,
furyl, indolizinyl,
indolyl, dihydroindolyl, imidazolyl, indazolyl, isobenzofuryl, isoindolyl,
isoindolinyl,
dihydroisoindolyl, isoquinolyl, dihydroisoquinolinyl, isoxazolyl,
isothiazolyl, oxazolyl,
oxadiazolyl, phenanthrolinyl, phenanthridinyl, purinyl, pyranyl, pyrazinyl,
pyrazolyl,
pyridyl, pyrim id inyl, pyridazinyl, pyrrolinyl,
pyrrolyl, pyrrolopyridinyl, quinolyl,
quinoxalinyl, quinazolinyl, tetrahydroquinolinyl,
tetrazolopyridazinyl,
tetrahydroisoquinolinyl, thiophenyl, thiazolyl, thiadiazolyl, thienopyridinyl,
thienyl,
thiophenyl, triazolyl, xanthenyl, and the like.
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[0131] The phenyl structure associated with some of the embodiments described
herein is depicted below. This structure can be unsubstituted, as shown below,
or can
be substituted such that a substituent can independently be in any position
normally
occupied by a hydrogen atom when the structure is unsubstituted. Unless a
point of
attachment is indicated by -I, attachment may occur at any position normally
occupied
by a hydrogen atom.
LJ
Phenyl
[0132] Each Ra can independently be H; optionally substituted hydrocarbyl;
optionally
substituted aryl, such as optionally substituted phenyl or optionally
substituted aryl;
optionally substituted heteroaryl, such as optionally substituted pyridinyl,
optionally
substituted furyl, optionally substituted thienyl, etc. In some embodiments,
each R8 can
independently be H, or Ci_12 alkyl, including: linear or branched alkyl such
as linear or
branched alkyl of the formula: CH3, C2H5, C3H7, C4H9, C5H11, C61-113, C7H15,
C9F117,
C9H19, C10H21, etc., or cycloalkyl of the formula: C3H5, C4H7, C5H9, C6H11,
C7H13, C8H15,
C9H17, CioH19, etc.
[0133] Pharmaceutical Compositions
[0134] According to other embodiments, the present disclosure provides for a
pharmaceutical composition including any one of the compounds described
herein.
[0135] Pharmaceutical compositions can be formed by combining a compound
disclosed herein, or a pharmaceutically acceptable prodrug or salt thereof,
with a
pharmaceutically acceptable carrier suitable for delivery to a subject in
accordance with
known methods of drug delivery. Accordingly, a "pharmaceutical composition"
includes
at least one compound disclosed herein together with one or more
pharmaceutically
acceptable carriers, excipients, or diluents, as appropriate for the chosen
mode of
administration.
[0136] A pharmaceutical composition including a compound of the disclosure can
be
formulated in a variety of forms depending upon the particular indication
being treated
and will be apparent to one of ordinary skill in the art. Formulating
pharmaceutical
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compositions including one or more compounds of the disclosure can employ
straightforward medicinal chemistry processes. The pharmaceutical compositions
can
be subjected to conventional pharmaceutical operations such as sterilization
and/or can
contain conventional adjuvants, such as buffering agents, preservatives,
isotonicifiers,
stabilizers, wetting agents, emulsifiers, etc.
[0137] The administration of the formulations of the present disclosure can be
performed in a variety of ways, including orally, subcutaneously,
intravenously,
intracerebrally, intranasally, transdermally,
intraperitoneally, intramuscularly,
intrapulmonary, intrathecally, vaginally, rectally, intraocularly, or in any
other acceptable
manner. The formulations can be administered continuously by infusion,
although bolus
injection is acceptable, using techniques known in the art, such as pumps
(e.g.,
subcutaneous osmotic pumps) or implantation. In some instances the
formulations can
be directly applied as a solution or spray,
[0138] An example of a pharmaceutical composition is a solution designed for
parenteral administration. Although in many cases pharmaceutical solution
formulations
are provided in liquid form, appropriate for immediate use, such parenteral
formulations
can also be provided in frozen or in lyophilized form. In the former case, the
composition
must be thawed prior to use. The latter form is often used to enhance the
stability of the
active compound contained in the composition under a wider variety of storage
conditions, as it is recognized by those of ordinary skill in the art that
lyophilized
preparations are generally more stable than their liquid counterparts. Such
lyophilized
preparations are reconstituted prior to use by the addition of one or more
suitable
pharmaceutically acceptable diluents such as sterile water for injection or
sterile
physiological saline solution.
[0139] Parenterals can be prepared for storage as lyophilized formulations or
aqueous
solutions by mixing, as appropriate, the compound having the desired degree of
purity
with one or more pharmaceutically acceptable carriers, excipients or
stabilizers typically
employed in the art (all of which are termed "excipients"), for example
buffering agents,
stabilizing agents, preservatives, isotonifiers, non-ionic detergents,
antioxidants and/or
other miscellaneous additives.
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[0140] Buffering agents help to maintain the pH in the range which
approximates
physiological conditions. They are typically present at a concentration
ranging from 2
mM to 50 mM of a pharmaceutical composition. Suitable buffering agents for use
with
the present disclosure include both organic and inorganic acids and salts
thereof, such
as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric
acid-trisodium
citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate
buffers (e.g.,
succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide
mixture,
succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g.,
tartaric acid-
sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric
acid-sodium
hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium
fumarate
mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-disodium
fumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodium
glyconate mixture,
gluconic acid-sodium hydroxide mixture, gluconic acid-potassium glyconate
mixture,
etc.), oxalate buffer (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-
sodium
hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate
buffers (e.g.,
lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture,
lactic acid-
potassium lactate mixture, etc.), and acetate buffers (e.g., acetic acid-
sodium acetate
mixture, acetic acid-sodium hydroxide mixture, etc.). Additional possibilities
are
phosphate buffers, histidine buffers, and trimethylamine salts such as Tris.
[0141] Preservatives can be added to retard microbial growth, and are
typically added
in amounts of 0.2%-1% (w/v). Suitable preservatives for use with the present
disclosure
include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben,
octadecyldimethylbenzyl ammonium chloride, benzalkonium halides (e.g.,
benzalkonium chloride, bromide or iodide), hexamethonium chloride, alkyl
parabens
such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-
pentanol.
[0142] lsotonicifiers can be added to ensure isotonicity of liquid
compositions and
include polyhydric sugar alcohols, preferably trihydric or higher sugar
alcohols, such as
glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol. Polyhydric
alcohols can be
present in an amount between 0.1% and 25% by weight, typically 1% to 5%,
taking into
account the relative amounts of the other ingredients.
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[0143] Stabilizers refer to a broad category of excipients which can range in
function
from a bulking agent to an additive which solubilizes the therapeutic agent or
helps to
prevent denaturation or adherence to the container wall. Typical stabilizers
can be
polyhydric sugar alcohols (enumerated above); amino acids such as arginine,
lysine,
glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-
phenylalanine,
glutamic acid, threonine, etc.; organic sugars or sugar alcohols, such as
lactose,
trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol,
galactitol, glycerol and
the like, including cyclitols such as inositol; polyethylene glycol; amino
acid polymers;
sulfur-containing reducing agents, such as urea, glutathione, thioctic acid,
sodium
thioglycolate, thioglycerol, alpha-monothioglycerol and sodium thiosulfate;
low
molecular weight polypeptides (i.e., <10 residues); proteins such as human
serum
albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic
polymers such
as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and
glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides
such as
raffinose, and polysaccharides such as dextran. Stabilizers are typically
present in the
range of from 0.1 to 10,000 parts by weight based on the active compound
weight.
[0144] Additional miscellaneous excipients include fillers (e.g., starch),
chelating
agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin
E), and
cosolvents.
[0145] Particular embodiments can include one or more of ethanol (<10%),
propylene
glycol (<40%), polyethylene glycol (PEG) 300 or 400 (<60%), N-N-
dimethylacetamide
(DMA, <30%), N-methyl-2-pyrrolidone (NMP, <20%), dimethyl sulfoxide (DMSO,
<20%)
co-solvents or the cyclodextrins (<40%) and have a pH of 3 to 9.
[0146] The compound(s) can also be entrapped in microcapsules prepared, for
example, by coascervation techniques or by interfacial polymerization, for
example
hydroxymethylcellulose, gelatin or poly-(methylmethacrylate) microcapsules, in
colloidal
drug delivery systems (for example liposomes, albumin microspheres,
microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such techniques are
disclosed
in Remington, The Science and Practice of Pharmacy, 21st Ed., published by
Lippincott
Williams & Wilkins, A Wolters Kluwer Company, 2005.
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[0147] Parenteral formulations to be used for in vivo administration generally
are
sterile. This is readily accomplished, for example, by filtration through
sterile filtration
membranes.
[0148] Generally, the pharmaceutical compositions can be made up in a solid
form
(including granules, powders, or suppositories) or in a liquid form (e.g.,
solutions,
suspensions, or emulsions). The compounds can be admixed with adjuvants such
as
lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic
acid, talc,
magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric
and
sulphuric acids, acacia, gelatin, sodium alginate, polyvinyl-pyrrolidine,
and/or polyvinyl
alcohol, and tableted or encapsulated for conventional administration.
Alternatively, they
can be dissolved in saline, water, polyethylene glycol, propylene glycol,
ethanol, oils
(such as corn oil, peanut oil, cottonseed oil, or sesame oil), tragacanth gum,
and/or
various buffers. Other adjuvants and modes of administration are well known in
the
pharmaceutical art. The carrier or diluent can include time delay material,
such as
glyceryl monostearate or glyceryi distearate alone or with a wax, or other
materials well
known in the art.
[0149] Oral administration of the compounds and compositions is one intended
practice of the disclosure. For oral administration, the pharmaceutical
composition can
be in solid or liquid form, e.g., in the form of a capsule, tablet, powder,
granule,
suspension, emulsion or solution. The pharmaceutical composition is preferably
made
in the form of a dosage unit containing a given amount of the active
ingredient. A
suitable daily dose for a human or other subject can vary widely depending on
the
condition of the subject and other factors, but can be determined by persons
of ordinary
skill in the art using routine methods.
[0150] Solid dosage forms for oral administration can include capsules,
tablets, pills,
powders, and granules. In such solid dosage forms, the active compound can be
admixed with at least one inert diluent such as sucrose, lactose, or starch.
Such dosage
forms can also include, as is normal practice, additional substances other
than inert
diluents, e.g., lubricating agents such as magnesium stearate. In the case of
capsules,
tablets, and pills, the dosage forms can also include buffering agents.
Tablets and pills
can additionally be prepared with enteric coatings. For buccal administration
the
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pharmaceutical compositions can take the form of tablets or lozenges
formulated in
conventional manners.
[0151] Liquid dosage forms for oral administration can include
pharmaceutically
acceptable emulsions, solutions, suspensions, syrups, and elixirs containing
inert
diluents commonly used in the art, such as water. Such pharmaceutical
compositions
can also include adjuvants, such as wetting, sweetening, flavoring, and
perfuming
agents.
[0152] The pharmaceutical compositions can be formulated for parenteral
administration by injection, e.g. by bolus injection, or infusion.
Formulations for injection
can be presented in unit dosage form, e.g. in glass ampoule or multi-dose
containers,
e.g. glass vials. The pharmaceutical compositions for injection can take such
forms as
suspensions, solutions, or emulsions in oily or aqueous vehicles, and can
contain
formulatory agents such as antioxidants, buffers, non-ionic detergents,
dispersants,
isotonicifiers, suspending agents, stabilizers, preservatives, dispersing
agents and/or
other miscellaneous additives.
[0153] Although in many cases pharmaceutical compositions provided in liquid
form
are appropriate for immediate use, such parenteral formulations can also be
provided in
frozen or in lyophilized form. In the former case, the pharmaceutical
composition must
be thawed prior to use. The latter form is often used to enhance the stability
of the
compound contained in the pharmaceutical composition under a wider variety of
storage
conditions, as it is recognized by those or ordinary skill in the art that
lyophilized
preparations are generally more stable than their liquid counterparts.
Parenterals can be
prepared for storage as lyophilized formulations by mixing, as appropriate,
the
compound having the desired degree of purity with one or more pharmaceutically
acceptable carriers, excipients, or stabilizers typically employed in the art
(all of which
are termed "excipients"), for example, antioxidants, buffers, non-ionic
detergents,
dispersants, isotonicifiers, suspending agents, stabilizers, preservatives,
dispersing
agents and/or other miscellaneous additives. Such lyophilized preparations are
reconstituted prior to use by the addition of one or more suitable
pharmaceutically
acceptable diluents such as sterile pyrogen-free water for injection or
sterile
physiological saline solution.
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[0154] For administration by inhalation (e.g., nasal or pulmonary), the
pharmaceutical
compositions can be conveniently delivered in the form of an aerosol spray,
from
pressurized packs or a nebulizer, and/or with the use of suitable propellant,
e.g.
dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane,
carbon
dioxide, or other suitable gases or mixture of gases.
[0155] The compounds or compositions can be admixed with adjuvants such as
lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic
acid, talc,
magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric
and
sulphuric acids, acacia, gelatin, sodium alginate, polyvinyl-pyrrolidine,
and/or polyvinyl
alcohol, and tableted or encapsulated for conventional administration.
Alternatively, they
can be dissolved in saline, water, polyethylene glycol, propylene glycol,
ethanol, oils
(such as corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth gum,
and/or
various buffers. Other adjuvants and modes of administration are known in the
pharmaceutical art. The carrier or diluent can include time delay material,
such as
glyceryl monostearate or glyceryl distearate alone or with a wax, or other
materials
known in the art.
[0156] In addition to the formulations described above, the pharmaceutical
compositions can also be formulated as depot preparations. Such long acting
formulations can be administered by implantation or by intramuscular
injection.
[0157] The compounds can also be entrapped in microcapsules prepared, for
example, by coascervation techniques or by interfacial polymerization, (for
example
hydroxymethylcellulose, gelatin or poly-(methylmethacylate) microcapsules), in
colloidal
drug delivery systems (for example liposomes, albumin microspheres,
microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such techniques are
disclosed
in Remington, The Science and Practice of Pharmacy, 21st Ed., published by
Lippincott
Williams & Wilkins, A Wolters Kluwer Company, 2005.
[0158] Suitable examples of sustained-release preparations include semi-
permeable
matrices of solid hydrophobic polymers containing the compound or composition,
the
matrices having a suitable form such as a film or microcapsules. Examples of
sustained-release matrices include polyesters, hydrogels (for example, poly(2-
hydroxyethyl-methacrylate) or poly(vinylalcohol)), polylactides, copolymers of
L-glutamic
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acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable
lactic
acid-glycolic acid copolymers such as the PROLEASE technology (Alkermes,
Cambridge, Massachusetts) or LUPRON DEPOT (injectable microspheres composed
of lactic acid-glycolic acid copolymer and leuprolide acetate; Abbott
Laboratories, Abbott
Park, Illinois), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-
vinyl acetate and lactic acid-glycolic acid enable release of molecules for
long periods
such as up to or over 100 days, certain hydrogels release compounds for
shorter time
periods.
[0159] Methods of Use
[0160] The pharmaceutical compositions disclosed herein can be used to treat a
viral
infection in a subject; wherein the viral infection is caused by a virus from
one the
following families: Arenaviridae, Arterivirus, Astroviridae, Birnaviridae,
Bromoviridae,
Bunyaviridae, Caliciviridae, Closteroviridae, Comoviridae, Coronaviridae,
Cystoviridae,
Filoviridae, Flaviviridae, Flexiviridae, Hepadnaviridae, Hepevirus,
Herpesviridae,
Leviviridae, Luteoviridae, Mesoniviridae, Mononegavirales, Mosaic Viruses,
Nidovirales,
Nodaviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae,
Picobirnaviridae,
Picobirnavirus, Picornaviridae, Potyviridae, Reoviridae, Retroviridae,
Roniviridae,
Sequiviridae, Tenuivirus, Togaviridae, Tombusviridae, Totiviridae, and
Tymoviridae,
[0161] According to more specific embodiments, the pharmaceutical compositions
can
be used to treat a viral infection caused by one or more of Alfuy virus, Banzi
virus,
bovine diarrhea virus, Chikungunya virus, Dengue virus (DNV),
Encephalomyocarditis
virus (EMCV), Hepatitis B virus (HBV), HCV, human cytomegalovirus (hCMV), HIV,
Ilheus virus, influenza virus (including avian and swine isolates),
rhinovirus, norovirus,
adenovirus, Japanese encephalitis virus, Kokobera virus, Kunjin virus,
Kyasanur forest
disease virus, louping-ill virus, measles virus, MERS-coronavirus (MERS),
metapneumovirus, any of the Mosaic Viruses, Murray Valley virus, parainfluenza
virus,
poliovirus, Powassan virus, respiratory syncytial virus (RSV), Rocio virus,
SARS-
coronavirus (SARS), St. Louis encephalitis virus, tick-borne encephalitis
virus, WNV,
Ebola virus, Nipah virus, Lassa virus, Tacaribe virus, Junin virus, and yellow
fever virus.
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[0162] In an embodiment, a method of treating a viral infection in a subject
can include
administering to the subject a therapeutically effective amount of a
pharmaceutical
composition having the structure
0
H
0
K.
In some cases, the viral infection is caused by Ebola virus.
[0163] Many RNA viruses share biochemical, regulatory, and signaling pathways.
These viruses include influenza viruses (including avian and swine isolates),
rhinovirus,
norovirus, DNV, RSV, WNV, HCV, parainfluenza virus, metapneumovirus,
Chikungunya
virus, SARS, MERS, poliovirus, measles virus, yellow fever virus, tick-borne
encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis virus,
Murray
Valley virus, Powassan virus, Rocio virus, louping-ill virus, Banzi virus,
Ilheus virus,
Kokobera virus, Kunjin virus, Alfuy virus, bovine diarrhea virus, any of the
Mosaic
Viruses, HIV, Ebola virus, Lassa virus, and the Kyasanur forest disease virus.
The
compounds, pharmaceutical compositions, and methods disclosed herein can be
used
to treat these viruses.
[0164] Antiviral activity against WNV, Nipah Virus, Lassa Fever Virus, and
Ebola Virus
in vitro is measured by focus-forming assay. Virus strains that are used in
these assays
include WNV-TX (WNV), WNV-MAD (WNV), NiV-Malaysia (Nipah), LASV-Josiah
(Lassa Fever), and ZEBOV-Mayinga (Ebola). Cultured human cells including human
umbilical vein cells (HUVEC) are seeded in tissue-culture plates and infected
with virus
at MOI of 0.01 to 0.5 for a duration including but not limited to 2 hours and
then
removed. Compound dilutions are prepared in 0.5% DMSO and used to treat cells
at
final concentrations of compound ranging 0.001 to 10 pM per well. Vehicle
control wells
contain 0.5% DMSO and are used to compare to drug treated cells. Virus
infections
after drug treatment are allowed to proceed for 48 to 96 hours. Virus
supernatants are
then harvested and used to infect new monolayer of permissive cells. The newly
infected cells are incubated overnight (18-24 hours) and used to measure the
level of
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infectious virus in the original supernatants by focus-forming assay using
methods
generally known in the art.
[0165] Methods disclosed herein include treating subjects (humans, mammals,
free-
range herds, veterinary animals (dogs, cats, reptiles, birds, etc.), farm
animals and
livestock (horses, cattle, goats, pigs, chickens, etc.), and research animals
(monkeys,
rats, mice, fish, etc.)) with pharmaceutical compositions disclosed herein.
Treating
subjects includes delivering therapeutically effective amounts.
Therapeutically effective
amounts include those that provide effective amounts, prophylactic treatments,
and/or
therapeutic treatments.
[0166] An "effective amount" is the amount of a compound necessary to result
in a
desired physiological change in the subject. Effective amounts are often
administered
for research purposes. Effective amounts disclosed herein reduce, control, or
eliminate
the presence or activity of viral infections and/or reduce, control, or
eliminate unwanted
side effects of viral infections. For example, an effective amount may result
in a
reduction in viral protein in a subject or assay, a reduction in viral RNA in
a subject or
assay, and/or a reduction in virus present in a cell culture.
[0167] A "prophylactic treatment" includes a treatment administered to a
subject who
does not display signs or symptoms of a viral infection or displays only early
signs or
symptoms of the viral infection such that treatment is administered for the
purpose of
diminishing, preventing, or decreasing the risk of developing the viral
infection further.
Thus, a prophylactic treatment functions as a preventative treatment against a
viral
infection. Prophylactic treatment may also include vaccines as described
elsewhere
herein. Prophylactic treatment may result in a lack of increase in viral
proteins or RNA in
a subject, and/or a lack of increase in clinical indicators of viral
infection, such as: loss
of appetite, fatigue, fever, muscle aches, nausea, and/or abdominal pain in
the case of
HCV; fever and/or headache in the case of WNV; and cough, congestion, fever,
sore
throat, and/or headache in the case of RSV. Prophylactic treatments can be
administered to any subject regardless of whether signs of viral infection are
present. In
some embodiments, prophylactic treatments can be administered before travel.
[0168] A "therapeutic treatment" includes a treatment administered to a
subject who
displays symptoms or signs of a viral infection and is administered to the
subject for the
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purpose of diminishing or eliminating the signs or symptoms of the viral
infection. The
therapeutic treatment can reduce, control, or eliminate the presence or
activity of
viruses and/or reduce, control, or eliminate side effects of viruses.
Therapeutic
treatment may result in a decrease in viral proteins or RNA in a subject,
and/or a
decrease in clinical indicators of viral infection, such as: loss of appetite,
fatigue, fever,
muscle aches, nausea, and/or abdominal pain in the case of HCV; fever and/or
headache in the case of WNV; and cough, congestion, fever, cyanosis, sore
throat,
and/or headache in the case of RSV.
[0169] For administration, therapeutically effective amounts (also referred to
herein as
doses) can be initially estimated based on results from in vitro assays and/or
animal
model studies. For example, a dose can be formulated in animal models to
achieve a
circulating concentration range that includes an IC50 as determined in cell
culture
against a particular target. Such information can be used to more accurately
determine
useful doses in subjects of interest.
[0170] The actual dose amount administered to a particular subject can be
determined
by a physician, veterinarian, or researcher taking into account parameters
such as
physical and physiological factors including target, body weight, severity of
condition,
type of viral infection, previous or concurrent therapeutic interventions,
idiopathy of the
subject, and route of administration.
[0171] Pharmaceutical compositions can be administered intravenously to a
subject for
treatment of viral infections in a clinically safe and effective manner,
including one or
more separate administrations of the composition. For example, 0.05 mg/kg to
5.0
mg/kg can be administered to a subject per day in one or more doses (e.g.,
doses of
0.05 mg/kg once-daily (QD), 0.10 mg/kg QD, 0.50 mg/kg QD, 1.0 mg/kg QD, 1.5
mg/kg
QD, 2.0 mg/kg QD, 2.5 mg/kg QD, 3.0 mg/kg QD, 0.75 mg/kg twice-daily (BID),
1.5
mg/kg BID or 2.0 mg/kg BID). For certain antiviral indications, the total
daily dose of a
compound can be 0.05 mg/kg to 3.0 mg/kg administered intravenously to a
subject one
to three times a day, including administration of total daily doses of 0.05-
3.0, 0.1-3.0,
0.5-3.0, 1.0-3.0, 1.5-3.0, 2.0-3.0, 2.5-3.0, and 0.5-3.0 mg/kg/day of
compounds of Table
1 using 60-minute QD, BID, or three times daily (TID) intravenous infusion
dosing. In
one particular example, antiviral pharmaceutical compositions can be
intravenously
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administered QD or BID to a subject with, e.gõ total daily doses of 1.5 mg/kg,
3.0
mg/kg, 4.0 mg/kg of a composition with up to 92-98% wt/wt of a compound of
Table 1.
[0172] Additional useful doses can often range from 0.1 to 5 pg/kg or from 0.5
to 1 pg
/kg. In other examples, a dose can include 1 pg /kg, 5 pg /kg, 10 pg /kg, 15
pg /kg, 20
pg /kg, 25 pg /kg, 30 pg /kg, 35 pg/kg, 40 pg/kg, 45 pg/kg, 50 pg/kg, 55
pg/kg, 60 pg/kg,
65 pg/kg, 70 pg/kg, 75 pg/kg, 80 pg/kg, 85 pg/kg, 90 pg/kg, 95 pg/kg, 100
pg/kg, 150
pg/kg, 200 pg/kg, 250 pg/kg, 350 pg/kg, 400 pg/kg, 450 pg/kg, 500 pg/kg, 550
pg/kg,
600 pg/kg, 650 pg/kg, 700 pg/kg, 750 pg/kg, 800 pg/kg, 850 pg/kg, 900 pg/kg,
950
pg/kg, 1000 pg/kg, 0.1 to 5 mg/kg, or from 0.5 to 1 mg/kg. In other examples,
a dose
can include 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30
mg/kg, 35
mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg,
75
mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 150 mg/kg, 200
mg/kg,
250 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg,
650
mg/kg, 700 mg/kg, 750 mg/kg, 800 mg/kg, 850 mg/kg, 900 mg/kg, 950 mg/kg, 1000
mg/kg, or more.
[0173] Therapeutically effective amounts can be achieved by administering
single or
multiple doses during the course of a treatment regimen (e.g., daily, every
other day,
every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks,
every 3
weeks, monthly, every 2 months, every 3 months, every 4 months, every 5
months,
every 6 months, every 7 months, every 8 months, every 9 months, every 10
months,
every 11 months, or yearly.
[0174] The administration of the pharmaceutical compositions of the present
disclosure
can be performed in a variety of ways, including orally, subcutaneously,
intravenously,
intracerebrally, intranasally, transdermally,
intraperitoneally, intramuscularly,
intrapulmonary, intrathecally, vaginally, rectally, intraocularly, or in any
other acceptable
manner. The pharmaceutical compositions can be administered continuously by
infusion, although bolus injection is acceptable, using techniques well known
in the art,
such as pumps (e.g, subcutaneous osmotic pumps) or implantation. In some
instances
the pharmaceutical compositions can be directly applied as a solution or
spray.
[0175] Particular embodiments provide for pharmaceutical compositions
including any
one or more of the compounds described herein, for the purpose of treating
and/or
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preventing disease in a subject. Additional embodiments provide for
pharmaceutical
compositions alone or in combination with an antigen. As such, in some
embodiments
the pharmaceutical compositions can be used as vaccines.
[0176] The disclosure provides for the use of the compounds as adjuvants.
[0177] The compounds, pharmaceutical compositions, and methods disclosed
herein
can be additive or synergistic with other therapies currently in development
or use. For
example, ribavirin and interferon-a provide an effective treatment for HCV
infection
when used in combination. Their efficacy in combination can exceed the
efficacy of
either drug product when used alone. The pharmaceutical compositions of the
disclosure can be administered alone or in combination or conjunction with
interferon,
ribavirin, and/or a variety of small molecules that are being developed
against both viral
targets (viral proteases, viral polymerase, and/or assembly of viral
replication
complexes) and host targets (host proteases required for viral processing,
host kinases
required for phosphorylation of viral targets such as NS5A, and inhibitors of
host factors
required to efficiently utilize the viral internal ribosome entry site, or
IRES).
[0178] The pharmaceutical compositions disclosed herein could be used in
combination or conjunction with adamantane inhibitors, neuraminidase
inhibitors, alpha
interferons, non-nucleoside or nucleoside polymerase inhibitors, NS5A
inhibitors,
antihistamines, protease inhibitors, helicase inhibitors, P7 inhibitors, entry
inhibitors,
IRES inhibitors, immune stimulators, HCV replication inhibitors, cyclophilin A
inhibitors,
A3 adenosine agonists, and/or microRNA suppressors.
[0179] Cytokines that could be administered in combination or conjunction with
the
pharmaceutical compositions disclosed herein include interleukin (IL)-2, IL-
12, IL-23, IL-
27, or IFN-y.
[0180] New HCV drugs that are, or will be, available for potential
administration in
combination or conjunction with the pharmaceutical compositions disclosed
herein
include ACH-1625 (Achillion); Glycosylated interferon (Alios Biopharma);
ANA598,
ANA773 (Anadys Pharm); ATI-0810 (Arisyn Therapeutics); AVL-181 (Avila
Therapeutics); LOCTERON (Biolex); CTS-1027 (Conatus); SD-101 (Dynavax
Technologies); Clemizole (Eiger Biopharmaceuticals); GS-9190 (Gilead
Sciences); Gl-
5005 (GlobalImmune BioPharma); Resiquimod / R-848 (Graceway Pharmaceuticals);
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Albinterferon alpha-2b (Human Genome Sciences); IDX-184, IDX-320, IDX-375
(Idenix); IM0-2125 (Idera Pharmaceuticals); INX-189 (Inhibitex); ITCA-638
(Intarcia
Therapeutics); ITMN-191/RG7227 (Intermune); ITX-5061, 1TX-4520 (iThen(
Pharmaceuticals); MB11362 (Metabasis Therapeutics); Bavituximab (Peregrine
Pharmaceuticals); PSI-7977, RG7128, PSI-938 (Pharmasset); PHX1766 (Phenomix);
Nitazoxanide / ALINIA8 (Romark Laboratories); SP-30 (Samaritan
Pharmaceuticals);
SCV-07 (SciClone); SCY-635 (Scynexis); TT-033 (Tacere Therapeutics);
Viramidine/taribavirin (Valeant Pharmaceuticals); Telaprevir, VCH-759, VCH-
916, VCH-
222, VX-500, VX-813 (Vertex Pharmaceuticals); and PEG-INF Lambda
(Zymogenetics).
[0181] New influenza and WNV drugs that are, or will be, available for
potential
administration in combination or conjunction with the pharmaceutical
compositions
disclosed herein include neuraminidase inhibitors (Peramivir, Laninamivir);
triple therapy
neuraminidase inhibitors, ribavirin, and amantadine (ADS-8902); polymerase
inhibitors (Favipiravir); reverse transcriptase inhibitor (ANX-201); inhaled
chitosan
(ANX-211); entry / binding inhibitors (Binding Site Mimetic, Flucide); entry
inhibitor,
(Fludase); fusion inhibitor, (MGAWN1 for WNV); host cell inhibitors
(lantibiotics);
cleavage of RNA genome (RNAi, RNAse L); immune stimulators (Interferon,
Alferon-
LDO; Neurokinin1 agonist, Homspera, Interferon Alferon N for WNV); and TG21.
[0182] Other drugs for treatment of influenza and/or hepatitis that are
available for
potential administration in combination or conjunction with the pharmaceutical
compositions include PEGinterferon alfa-2a (Pegasys), PEGinterferon alfa-2b
(Peg-
Intron), ribavirin (Copegus; Rebetol), oseltamivir (Tamiflu), zanamivir
(Relenza),
amantadine, and rimantadine.
[0183] These agents can be incorporated as part of the same pharmaceutical
composition or can be administered separately from the compounds of the
disclosure,
either concurrently or in accordance with another treatment schedule.
[0184] The compounds or pharmaceutical compositions can be additive or
synergistic
with other compounds or pharmaceutical compositions to enable vaccine
development.
By virtue of their antiviral and immune enhancing properties, the compounds
can be
used to affect a prophylactic or therapeutic vaccination. The compounds need
not be
administered simultaneously or in combination with other vaccine components to
be
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effective. The vaccine applications of the compounds are not limited to the
treatment of
viral infection but can encompass all therapeutic and prophylactic vaccine
applications
due to the general nature of the immune response elicited by the compounds,
[0185] A "vaccine" is an immunogenic preparation that is used to induce an
immune
response in an individual. A vaccine can have more than one constituent that
is
immunogenic. A vaccine can be used for prophylactic and/or therapeutic
purposes. A
vaccine does not necessarily have to prevent viral infections. Without being
bound by
theory, the vaccines of the disclosure can affect an individual's immune
response in a
manner such that viral infection occurs in a lesser amount (including not at
all) or such
that biological or physiological effects of the viral infection are
ameliorated when the
vaccine is administered as described herein. As used herein, vaccines include
preparations including pharmaceutical compositions including the compounds,
alone or
in combination with an antigen, for the purpose of treating a viral infection
in a subject
including a vertebrate animal.
[0186] The disclosure provides for the use of the compounds and pharmaceutical
compositions as adjuvants. An adjuvant enhances, potentiates, and/or
accelerates the
beneficial effects of another administered therapeutic agent. In particular
embodiments,
the term "adjuvant" refers to compounds that modify the effect of other agents
on the
immune system. Adjuvants that possess this function may also be inorganic or
organic
chemicals, macromolecules, or entire cells of certain killed bacteria, which
enhance the
immune response to an antigen. They may be included in a vaccine to enhance
the
recipient's immune response to the supplied antigen.
[0187] As is understood by one of ordinary skill in the art, vaccines can be
against
viruses, bacterial infections, cancers, etc. and can include one or more of a
live
attenuated vaccine (LAIV), an inactivated vaccine (IIV; killed virus vaccine),
a subunit
(split vaccine); a sub-virion vaccine; a purified protein vaccine; or a DNA
vaccine.
Appropriate adjuvants include one or more of water/oil emulsions, non-ionic
copolymer
adjuvants, e.g., CRL 1005 (Optivax; Vaxcel Inc., Norcross, Ga.), aluminum
phosphate,
aluminum hydroxide, aqueous suspensions of aluminum and magnesium hydroxides,
bacterial endotoxins, polynucleotides, polyelectrolytes, lipophilic adjuvants
and synthetic
muramyl dipeptide (norMDP) analogs such as N-acetyl-nor-muranyl-L-alanyl-D-
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isoglutamine, N-acetyl-muranyl-(6-0-stearoyI)-L-alanyl-D-isoglutamine, or N-
Glycol-
muranyl-LalphaAbu-D-isoglutamine (Ciba-Geigy Ltd.).
[0188] The present disclosure further includes the use and application of the
compounds and pharmaceutical compositions in vitro in a number of applications
including developing therapies and vaccines against viral infections, research
in
modulation of the innate immune response in eukaryotic cells, etc. The
compounds and
pharmaceutical compositions disclosure can also be used in animal models. The
results
of such in vitro and animal in vivo uses of the compounds and pharmaceutical
compositions can, for example, inform their in vivo use in humans, or they can
be
valuable independent of any human therapeutic or prophylactic use.
[0189] The Examples below are included to demonstrate particular embodiments
of
the disclosure. Those of ordinary skill in the art should recognize in light
of the present
disclosure that many changes can be made to the specific embodiments disclosed
herein and still obtain a like or similar result without departing from the
spirit and scope
of the disclosure. For example, the Examples below provide in vitro methods
for testing
the compounds of the disclosure. Other in vitro and/or in vivo virus infection
models
include flaviviruses such as DNV, bovine diarrheal virus, WNV, and GBV-C
virus, other
RNA viruses such as RSV, SARS, and the HCV replicon systems. Furthermore, any
appropriate cultured cell competent for viral replication can be utilized in
the antiviral
assays.
[0190] EXPERIMENTAL EXAMPLES
[0191] Example 1. General Synthesis methods.
[0192] The compounds of the disclosure may be prepared by the methods
described
below, together with synthetic methods familiar to those of ordinary skill in
the art. The
starting materials used herein are commercially available or may be prepared
by routine
methods known in the art (such as those methods disclosed in standard
reference
books such as the COMPENDIUM OF ORGANIC SYNTHETIC METHODS, Vol. I-VI
(published by Wiley-Interscience)). Preferred methods include those described
below.
[0193] During any of the following synthetic sequences it may be necessary
and/or
desirable to protect sensitive or reactive groups on any of the molecules
concerned.
This can be achieved by means of conventional protecting groups, such as those
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described in T. W. Greene, Protective Groups in Organic Chemistry, John Wiley
&
Sons, 1981; T. W. Greene and P. G. M. Wuts, Protective Groups in Organic
Chemistry,
John Wiley & Sons, 1991, and T. W, Greene and P. G. M. Wuts, Protective Groups
in
Organic Chemistry, John Wiley & Sons, 1999.
[0194] Compounds of the disclosure, or their pharmaceutically acceptable
salts, can
be prepared according to the reaction Schemes described below. These methods
can
be modified or adapted in ways known to chemists of ordinary skill in order to
achieve
synthesis of additional compounds within the scope of the present invention.
Such
modification was done to synthesize an exemplary compound of the invention as
described in Examples 2 and 3. Unless otherwise indicated, the substituents in
the
Schemes are defined as above. Isolation and purification of the products is
accomplished by standard procedures, which are known to a chemist of ordinary
skill.
[0195] It will be understood by one skilled in the art that the various
symbols,
superscripts and subscripts used in the schemes, methods and examples are used
for
convenience of representation and/or to reflect the order in which they are
introduced in
the schemes, and are not intended to necessarily correspond to the symbols,
superscripts or subscripts in the appended claims. The schemes are
representative of
methods useful in synthesizing the compounds of the present invention. They
are not to
constrain the scope of the disclosure in any way.
[0196] Synthesis of N-(4-hydroxy-2-methyl-1,3-benzothiazol-5-ypacetamide:
NoNH
-FLO
0.6 g of commercially available 5-azido-2-methyl-1 ,3-benzothiazole and 5 g of
acetic
acid were heated to 100 C for 20 minutes. Evaporation and column
chromatography
purification of the residue afforded 0.43 g of N-(4-hydroxy-2-methyl-1,3-
benzothiazol-5-
yl)acetamide.
[0197] Synthesis of 5-amino-2-methyl-1,3-benzothiazol-4-ol:
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\
SOH S ...,.... OH
/ ----0- 1
io
t
..õ,....
0
0.4 g of the acetamide was treated with 2 mL concentrated HCI. Evaporation
provided
0.38 g of 5-amino-2-methyl-1,3-benzothiazol-4-ol as the di-HCl salt.
[0198] Synthesis of 7-methyl[1,3]thiazolo[5,4-g][1,3]benzoxazol-2-amine:
S OH
)...)-.1
"N> NH2
To a solution of 5 mL 3:4 methanol:water solution cooled to 0 0C was added
0.08 mL
bromine followed by 0.12 g of KCN in portions. When the bromine color was gone
the
cyanogen bromide solution was added to 0.38 g of the amine dihydrochloride in
20 mL
water and 0.252 g sodium bicarbonate and the reaction was left overnight. The
reaction
was filtered and the filtrate was treated with sodium bicarbonate and
concentrated
under vacuum. The residue was dissolved in ethanol and the solution was
filtered. The
filtrate was concentrated to a residue which was purified by chromatography to
afford
0.14 of 7-methyl[1,3]thiazolo[5,4-01,3]benzoxazol-2-amine.
[0199] Example 2. Synthesis of N-(7-methyl[1,3]thiazolo[5,4-g][1,3]benzoxazol-
2-
yl)thiophene-2-carboxamide
[0200] Acid chloride coupling was performed:
.0
¨ />--- NH2
To a suspension of 0.15 g 7-methyl[1,3]thiazolo[5,4-01,3ibenzoxazol-2-amine in
2.5
mL dry pyridine was added 0.078 mL thiophene-2-carbonyl chloride. The reaction
was
stirred for 5 h at 80 C then cooled to room temperature. 4 mL of water was
added and
the precipitate was filtered off, washed with water and dried to afford 0.154
g of N-(7-
methyl[1,3]thiazolo[5,4-g][1,3]benzoxazol-2-yl)thiophene-2-carboxamide.
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[0201] Example 3. Synthesis of
N - [6 - (pyrrolidine - 1 - sulfonyl) - 1,3 - benzothiazol - 2 -
yl]naphthalene - 2 - carboxa
mide
[0202] The intermediate 6-(pyrrolidin-1-ylsulfony1)-1,3-benzothiazol-2-amine,
which
was used in the synthesis of
N - [6 - (pyrrolidine - 1 - sulfonyl) - 1,3 - benzothiazol - 2 -
yl]naphthalene - 2 - carboxa
mide, was synthesized as described below:
CI 8
CI8
0 s
0 -10- s N H2
N H2 N
A mixture of commercially available 4-(pyrrolidin-1-ylsulfonyl)aniline (1.0 g)
and
ammonium thiocyanate (1.01g) was suspended in 25 mL acetic acid and heated to
90
C. The mixture was cooled to 15 C and liquid bromine (0.22 mL) was added
dropwise.
The reaction was stirred at room temperature overnight then filtered. The
filtrate was
concentrated under vacuum and the residue was added to a solution of aqueous
sodium bicarbonate and stirred for 1 hour. The precipitate was filtered off,
washed with
water and ether and dried to afford 0.7 g of 6-(pyrrolidin-1-ylsulfonyI)-1,3-
benzothiazol-
2-am ine.
[0203] 0.1g of 6-(pyrrolidin-1-ylsulfony1)-1,3-benzothiazol-2-amine was
dissolved in 2
mL dry pyridine and 2-naphthoyl chloride (0.067 g) was added and the mixture
was
stirred at 80 C for 5 h. After cooling to room temperature the mixture was
added to 0.7
mL water and the precipitate was filtered off, washed with water and ether and
dried to
afford 0.091 g of
N - [6 - (pyrrolidine - 1 - sulfonyl) - 1,3 - benzothiazol - 2 -
yl]naphthalene - 2 - carboxa
mide:
o .11
0
C\I I/ ici)
S
zis 0 s>_
s 0
NH2 -OP-
N
N
64
SUBSTITUTE SHEET (RULE 26)
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Example 4. antiviral activity and pharmacological properties using structure-
activity
relationship (SAR) studies
[0204] This Example describes optimization of compounds for antiviral action.
First, a
small analog derivative set is used to define a structural class. The active
analogs that
are identified in this first stage are then used to define a subset of
structural classes of
interest for further optimization (Stage 2).
[0205] Stage 2 focuses on creating structural diversity and evaluating core
variants for
derivative expansion. Structural derivatives are tested for biological
activity including the
IRF-3 translocation assay, antiviral activity, and cytotoxicity in one or more
cell lines or
peripheral blood mononuclear cells. Optimized molecules that show improved
efficacy
and low cytotoxicity are further characterized by additional measures of in
vitro
toxicology and absorption, distribution, metabolism, and elimination (ADME).
Their
mechanism of action and breadth of antiviral activity are also studied.
[0206] To design analog structures, the drug-like properties, metabolic
lability, and
toxic potential of the lead compounds are analyzed. Drug-like properties, as
measured
by Lipinski's Rules, and related physiochemical properties are primary
indicators of
bioavailability. Structural features that suggest metabolic and toxicological
liabilities may
indicate limited stability, reduced half-life, reactive intermediates, or
idiosyncratic toxicity
and will therefore be removed.
[0207] Compounds are tested for potent in vitro antiviral activity against
viruses
including HCV 2A, RSV, DNV type 2, and influenza A virus strains. Viral
protein and
RNA levels are assessed following drug treatment using the assays described
herein.
Analog design is aimed to identify lead compounds with picomolar to nanomolar
potency, which is adequate to support preclinical development Lead compounds
are
characterized for their in vitro toxicological and ADME properties and for
further
mechanistic study.
[0208] In vitro pharmacology studies are performed to measure performance of
the
most promising analogs in one or more assays of intestinal permeability,
metabolic
stability and toxicity. Key in vitro characterization studies can include
plasma protein
binding; serum, plasma, and whole-blood stability in human and model
organisms;
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intestinal permeability; intrinsic clearance; human Ether-à-go-go (hERG)
channel
inhibition; and genotoxicity.
[0209] For each analog, an HPLC- and/or HPLC-mass spectrometry-based
analytical
method is used to evaluate drug and metabolite concentrations in various test
systems.
Although the specific analytical method is optimized for each molecule,
reverse-phase
chromatography can be used alone or in combination with quadrupole mass
spectrometry to characterize the identity and purity of several of the lead
molecules.
Initially, drug stability over time in increasing concentrations of serum,
plasma, and
whole blood from mammalian species (such as mouse, cynomolgus macaque, and
human) is evaluated by HPLC, and a half-life is determined.
[0210] Prominent metabolites were characterized by mass spectrometry. Human
plasma protein binding were evaluated by partition analysis using equilibrium
dialysis.
For intestinal permeability modeling, apical-to-basolateral flux is assessed
in the human
epithelial cell line TC7. Hepatic clearance is estimated for a subset of the
most
promising analogs by measuring the rate of disappearance of the parent
compound
during incubation in human liver microsomes. As above, specific metabolites
can be
isolated and characterized.
[0211] In vitro toxicology studies are performed to evaluate the potential
cardiac and
genetic toxicity of lead analogs. Automated patch-clamp is used to assess the
impact of
each compound on hERG channel currents in a recombinant Chinese hamster ovary
(CHO) cell line transgenically expressing the human Kv11.1 gene.
Concentrations up to
the lesser of 30 times the maximum serum concentration or the limit of
solubility of each
compound are evaluated in order to determine an IC50 for the molecule on the
hERG
channel. A subset of compounds is evaluated over a range of concentrations for
their
ability to induce mutation reversion in Salmonella typhimurium strains TA98
and TA100
or to promote micronucleus formation in CHO cells in culture.
[0212] Example 5. Biological activity.
[0213] This Example describes methods used to identify compounds that activate
innate immune responses, including activation of the RIG-I pathway. Other
compounds
as described herein likewise can be evaluated by the methods described in this
example, and other cell types can also be used.
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[0214] Cultured Huh 7 cells that were stably transfected with a luciferase
reporter gene
coupled with a RIG-I signaling pathway responsive promoter (IFNp, ISG56, or
ISG54
promoter) were seeded and allowed to grow overnight. The compound 1 was then
added and cells were grown in the presence of compound 1 for 18-20 hours.
Steady-
Glo luciferase substrate (Promega) was added and luminescence was read on a
luminometer (Berthold).
[0215] Figure 1A shows that compound 1 of Table 1 as described herein was
validated
by demonstrating dose-dependent induction of the luciferase reporter gene
coupled to
the promoters for IFNP ("IFN13-LUC," left), ISG56 ("ISG56-LUC," center), and
ISG54
("ISG54-LUC," right). Additionally, compound 1 did not induce a nonspecific
promoter
(13-actin-LUC, Figure 1B).
[0216] An immunofluorescent cytochemistry assay was used to determine IRF-3
activation and translocation to the nucleus. Cultured human HeLa cells were
treated
with increasing amounts of compound or equivalent amounts of DMSO diluted in
media
for 20 hours. Positive control wells were infected with 100 HA/mL Sendai virus
for an
equivalent time period. IRF-3 was detected using polyclonal rabbit serum
specific to
IRF-3 and a secondary antibody conjugated to DYLIGHT (Pierce Biotechnology,
Inc.,
Rockford, IL) 488.
[0217] An immunofluorescent cytochemistry assay was used to determine NFKB
activation. The innate immune response is also dependent on activation of the
NFKB
transcription factor. Cultured human HeLa cells were treated with increasing
amounts of
compound or equivalent amounts of DMSO diluted in media for 20 hours. Positive
control wells were infected with 100 HA/mL Sendai virus for an equivalent time
period.
NFKB was detected using monoclonal mouse antibody specific to the p65 subunit
of
NFKB and a secondary antibody conjugated to DyLight 488.
[0218] Quantification of the IRF-3 and NFKB immunofluorescent assays described
herein was done as follows: 96-well plates containing cultured human cells
treated with
compound and stained for either IRF-3 or NFKB were scanned and quantified
using the
ARRAYSCAN instrument and software (Cellomics). Activation of transcription
factor
was evidenced by increased nuclear intensity normalized for cytoplasmic
intensity, or
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nuclear-cytoplasmic difference. Compound 1 shows a dose dependent increase in
nuclear-cytoplasmic difference for IRF-3 (Figure 1C) and for NFKB (Figure 1D).
[0219] Assays for innate immune gene expression were performed in cell types
including HeLa cells, PH5CH8 cells, and HUVEC primary cells. Gene expression
can be
similarly assayed in cell types that include: primary blood mononuclear cells,
human
macrophages, THP-1 cells, Huh 7 cells, A549 cells, MRC5 cells, rat
splenocytes, rat
thymocytes, mouse macrophages, mouse splenocytes, and mouse thymocytes.
Expression of other genes of interest can be assayed as described herein.
[0220] Cultured HeLa cells were treated with 20pM, 10pM, 5pM of compound or a
DMSO control and incubated for up to 24 hours. Cultured PH5CH8 cells were
treated
with 1 OpM, 5pM, 1pM, or a DMSO control and incubated for up to 24 hours.
Primary
HUVEC cells were thawed and seeded in 6-well plates at 2.4x104 cells per well
and
allowed to grow to 80% confluence, typically 5 days in culture with fresh
media replaced
every 48 hours. Compound was added at 10pM, 1pM or a DMSO control and
incubated
for up to 24 hours. Gene expression was assayed as described below.
[0221] Cells were harvested and RNA was isolated using the QIAshredder columns
and RNeasy Mini Kit (Qiagen) according to manufacturer instructions. Reverse
transcription was performed and the cDNA template was used for quantitative
real-time
PCR. PCR reactions were performed using commercially available, validated
TaqMan
gene expression assays (Applied Biosystemsitife Technologies) according to
manufacturer instructions. Gene expression levels were measured using a
relative
expression analysis (AACt).
[0222] Figures 2A-2C show induction of gene expression by compounds 1 and 2 of
Table 1. Figure 2A shows gene expression levels of IFIT2 (left) and OAS1
(right) in
HeLa cells over time from 4-24 hours post treatment with 10pM compound 1
(grey;
OAS1 only) or 1 OpM compound 2 (black; IFIT2 and OAS1 both shown). Figure 2B
shows gene expression levels of IFIT2 in PH5CH8 cells (solid color bars) and
HeLa
cells (black checked bars) treated with 1 OpM compound 1 (CPD 1) or compound 2
(CPD 2). Figure 2C shows gene expression levels of IFIT2 (left), OAS1
(center), and
MxA (right) in primary HUVEC cells that were treated with 1pM compound 1 (CPD
1) or
1pM compound 2 (CPD 2).
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[0223] Figures 3A-3B show induction of gene expression by compound 3 and
compound 7 of Table 1, Figure 3A shows IFIT2 gene expression was induced by
5pM
compound 3 or compound 7. Figure 3B shows compound 3 induced innate immune
gene expression in mouse macrophage cells.
[0224] Example 6. Ex vivo immune stimulatory activity of compound 1
[0225] The activity of compound 1 of Table 1 in primary immune cells was
assayed to
determine whether compound 1 stimulates immune responses. Cultured human
primary
dendritic cells were treated with 0,1, or 10 pM of compound 1 for 24 hours.
Supernatant
from treated wells was isolated and tested for levels of cytokine protein.
Cytokines were
detected using specific antibodies conjugated to magnetic beads and a
secondary
antibody that reacts with Streptavidin/Phycoerythrin to produce a fluorescent
signal. The
bound beads were detected and quantified using the MAGPIV) (Luminex Corp.)
instrument, although similar techniques as are known in the art may be used to
measure fluorescent protein production, such as for example an ELISA,
[0226] Figure 4 shows induction of the chemokines IL-8, MCP-1, MIP-1a, and MIP-
113
by dendritic cells treated with compound 1 of Table 1 (concentrations shown in
pM).
LPS is shown as a positive control inducer of chemokine expression.
[0227] Other cells from which cytokine secretion can be measured include, for
example human peripheral blood mononuclear cells, human macrophages, mouse
macrophages, mouse splenocytes, rat thymocytes, and rat splenocytes.
[0228] Example 7. In vitro antiviral activity.
[0229] To further characterize the breadth of antiviral activity of optimized
molecules,
cell culture infection models are used to analyze different viruses, including
different
strains of influenza virus, HCV, DNV, RSV, and WNV, an emerging public health
concern. The studies include treating cells with compound 2-24 hours prior to
infection
or treating cells up to 8 hours after infection. Virus production and cellular
ISG
expression are assessed over a time course to analyze antiviral effects of
representative compounds from lead structural classes. Known antiviral
treatments
including IFNii are used as a positive control.
[0230] Virus production is measured by focus-forming or plaque assay. In
parallel
experiments, viral RNA and cellular ISG expression are measured by qPCR and
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immunoblot analyses. These experiments are designed to validate compound
signaling
actions during virus infection, and assess compound actions to direct innate
immune
antiviral programs against various strains of viruses and in the setting of
virus
countermeasures. Detailed dose-response analyses of each compound are
conducted
in each virus infection system to determine the effective dose that suppresses
virus
production by 50% (IC50) and 90% (1C90) as compared with control cells for
both the
pre-treatment and post-treatment infection models.
[0231] Compounds of the current invention are tested using in vitro models
against
viruses including: Hepatitis B Virus (HBV), HCV H77 (genotype 1a), HVV JFH1
(genotype 2a), Influenza A/PR/8/34 (H1N1 mouse-adapted virus), Influenza
ANVSN/33
(H1N1 mouse-adapted neurovirulent virus), Influenza AfTX/36/91 (H1N1
circulating
virus), Influenza A/Udorn/72 (H3N2), WNV TX02 (lineage 1), WNV MAD78 (lineage
2),
RSV, human coronavirus 0C43 (SARS-like pathogen), and DNV type 2.
[0232] Antiviral activity of exemplary compounds of the disclosure is
demonstrated in
Examples 8-11 below.
[0233] Example 8. In vitro activity against respiratory syncytial virus
[0234] HeLa cells were seeded the previous day in 6-well plates at 4x1O5 cells
per well.
The next day, the media was replaced with RSV in media without FBS at an MO1
of 0.1.
Virus binding occurred at 37 C for 2 hours. After 2 hours the cells were
washed with
warm complete media and replaced with media containing drug at varying
concentrations of lOpM, 5pM, 1pM, or a DMSO control. Cells were placed in a 37
C
incubator for 48 hours.
[0235] For virus detection and titration, HeLa cells were seeded in 96-well
plates at
8x103 cells per well 24hrs prior to collecting virus supernatant. After the 48
hour
incubation period, the virus supernatant from the infected plate was harvested
and used
to infect these cells at a 1/10 final dilution. Cells were placed in a 37 C
incubator for 24
hours.
[0236] 24 hours after infection, cells were washed twice with PBS and fixed
with
methanol/acetone solution. After fixing the cells were washed twice with PBS
and
replaced with blocking buffer (10% horse serum, 1g/rnt.. BSA and 0.1% Triton-
100X in
PBS) for 1 hour. The blocking buffer was replaced with binding buffer
containing a
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1/2000 dilution of primary antibody for 2 hours at room temperature. The
primary
antibody was a mouse monoclonal antibody against RSV. The cells were washed
twice
with PBS and replaced with binding buffer containing 1/3000 dilution of the
Alexa Fluor-
488 goat anti-mouse secondary antibody and a Hoechst nuclear stain for 1 hour
at room
temperature. The cells were washed twice with PBS and PBS is added to all
wells. The
96-well plate was sealed and fluorescence activity associated with virus
infectivity was
determined by immunofluorescent assay using the Array Scan instrument (Thermo-
Fischer).
[0237] Treatment with compounds can be done prior to infection. In variations
of this
method, the compounds are added at varying time points prior to infection with
virus.
Virus detection and titration is conducted as described.
[0238] Figure 5 shows results of experiments performed using the protocol of
Example
8, demonstrating the antiviral activity of select compounds that demonstrated
antiviral
activity against RSV. ++4- = greater than 70% inhibition of infection, ++ =
greater than
50% inhibition, + = greater than 30% inhibition, - = less than 30% inhibition.
Example 9. In vitro activity against influenza virus.
[0239] Influenza A/Worn/72 infection of H292 cells. 2x106 H292 cells in
RPMI1640+10%FCS were treated with 2pM compound 2 in a final concentration of
0.5% DMSO for 6 hours. Compound-containing media was aspirated and replaced
with
1X MEM containing A/Udorn/72 at an MOI of 0.1 and placed at 37 C in a CO2
incubator.
Two hours post infection, virus-containing media was aspirated and replaced
with 1X
MEM containing 1 ug/ml_ TPCK-treated Trypsin, 2pM compound 2, 0.5% DMSO. Cells
were placed in 37 C CO2 incubator for 18 hours. After 20 hours post-infection,
virus
supernatants were collected and titered on MDCK cells.
[0240] Influenza A/Udorn/72 infection of HEK293 cells. 5x105 HEK293 cells were
infected with A/Udorn/72 at an MOI of 0.2 in 1X MEM. After 2 hours post-
infection,
virus-containing media was aspirated and replaced with 1X MEM containing 1
pg/mL
TPCK-treated Trypsin, 1 OpM compound 2, 0.5% DMSO. Cells were returned to 37
C,
CO2 incubator for 18 hours. After 20 hours post-infection, virus supernatants
were
collected and titred on MDCK cells.
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[0241] Titer in MDCK cells. 10pL of infected supernatant was added to 2x106
MDCK
cells in the presence of 2pg/mL TPCK-trypsin and placed in a 37 C CO2
incubator.
After 8 hours, supernatant was removed and cells were fixed and stained with
FITC-
conjugated antibody specific for Influenza NP protein. Number of foci was
quantitated
using the ArrayScan instrument and software (Cellomics).
[0242] The protocol of Example 9 can be performed as described in order to
evaluate
the antiviral activity of example compounds. Figure 6 shows antiviral activity
of example
compounds against Influenza A virus Udorn/72. Treatment of HEK293 cells with
increasing concentrations of compound 3, compound 7, compound 9, and compound
10
of Table 1 resulted in dose-dependent inhibition of virus infection (shown as
%
untreated negative control), Calculated IC50 values are shown. Table 2 shows
calculated IC50 values of selected compounds from Table 1.
Table 2. 1050 against flu
____________________________________________________ 1050 (i.J1V1)
Compound
2 2.04
________________ 3 0.61
4 _____________________________________________________ 1.29
,, 5 ................................... >10 __
, 1.49
________________ 6
________________ 7 >5
9 ______________________________________________________ 1.15
3.18 ¨ ______________________________________________________________________
_1101
______516
------------------------------------------------------ 12 2
13 ____________________________________________________ 2
________________ 15 6.8 __
[0243] Example 10, In vitro activity against Dengue virus.
[0244] Cultured human Huh 7 cells were seeded in 6-well tissue-culture plates
at a
density of 4x105 cells per well and grown for 24 hours. Cells were infected
with DNV
type 2 strain at multiplicity of infection (M01) of 0.1 for 2 hours and then
removed.
Compound dilutions were prepared in 0.5% DMSO and used to treat cells at final
concentrations of compound ranging 0.001 to 10 pM per well. Vehicle control
wells
treated with 0,5% DMSO were used to compare to drug treated cells. Replication
was
allowed to proceed for 48 hours. Virus supernatants were harvested and used to
infect
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new monolayer of permissive cells, such as Vero cells that were seeded in 96-
well
plates at 8x103 cells per well 24hrs prior to collecting virus supernatant.
[0245] The newly infected cells were incubated for 24 hours and used to
measure the
level of infectious virus in the original supernatants by immunofluorescent
staining of
viral protein. The cells were fixed with ice-cold 11 methanol and acetone
solution and
stained for DNV fusion protein. Primary mouse monoclonal antibody against DNV
fusion
protein (Millipore) was used at 1:2000 dilution. Secondary goat anti-mouse
antibody
conjugated to Alexa Fluor 488 dye (Invitrogen) and Hoescht Dye (nuclear
staining) are
used at 1:3000 to detect DNV protein and cell nuclei. Following secondary
antibody
incubation, the monolayers were washed and left in 100pL PBS for imaging and
quantitation using a Cellomics ArrayScan HCS instrument.
[0246] Figure 7 shows the antiviral activity of compound 5 and compound 20 of
Table
1 against Dengue virus (DNV) type 2. Treatment with increasing amounts of
compound
showed dose-dependent decrease in infection by virus.
[0247] Figure 8 shows antiviral activity of exemplary compounds against Dengue
virus
type 2. Treatment of Huh 7 cells with increasing concentrations of compound 8,
compound 3, compound 5, compound 6, compound 7, compound 9, and compound 10
of Table 1 resulted in dose-dependent inhibition of virus infection (shown as
A)
untreated negative control). Calculated IC50 values are shown.
[0248] Table 3 shows calculated IC50 values of selected compounds against
Dengue
virus type 2 (DV2) and/or Dengue virus type 4 (DV4).
Table 3. IC50 against DNV
[1 _____ Compound
õ.
' IC50 against DV2 (pM) --
. ..
1.87 ... -4-- IC50 against DV4 (pM)
2 6.18
_I
-
_____________________________________________________________________________
3 ______________________________________________________________ 0.78
______ i
,,, ....1
4 3.65
, __
0.47 4.9
2.03
6 0.02
'
- .............................................
___________ 7 0.74 __________________________________________ 1.87
........... 8 1.23
_
___________ 9 1.78 __
............................ ........_ .......... _. _________
___________ 10 1.78 __________________________________________ 6.48
'
11 0.53
21 0.10 _________________ 0.27
_ ...
12 _____________________________________ 0.14 .... 1 0.15 __
.. _
___________ 14 i 0.15 ______________ 0.03
i
................................................... ., _____________________
____
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__________ 15 0.39 >5 __
16 __________________________________ >5
17 0.50 ____________________ 4.7 .......
ji
[0249] Example 11. In vitro activity against human coronavirus.
[0250] MRC5 cells were seeded the previous day in 6-well plates and grown for
24
hours. Cells were infected with human coronavirus 0C43 (HCoV-0C43) for 2 hours
and
then removed. Compound dilutions were prepared in 0.5% DMSO and used to treat
cells at final concentrations of compound ranging 0.001 to 10 pM per well.
Vehicle
control wells treated with 0.5% DMSO were used to compare to drug treated
cells.
Replication was allowed to proceed for 5 days. Virus supernatants were
harvested and
used to infect new monolayer of permissive cells, such as Huh 7 cells that
were seeded
in 96-well plates 24 hours prior to collecting virus supernatant, i.e., 4 days
post infection.
[0251] The newly infected cells were incubated for 48 hours and used to
measure the
level of infectious virus in the original supernatants by immunofluorescent
staining of
viral protein. The cells are fixed with ice-cold 1:1 methanol and acetone
solution and
stained for HCoV-0C43 nucleoprotein. Primary mouse monoclonal antibody against
HCoV-0C43 nucleoprotein (Millipore) is used at 1:1000 dilution. Secondary goat
anti-
mouse antibody conjugated to Alexa Fluor 488 dye (Invitrogen) and Hoescht Dye
(nuclear staining) were used at 1:3000 to detect 0C43 protein and cell nuclei.
Following
secondary antibody incubation, the monolayers were washed and left in 100pL
PBS for
imaging and quantitation using a Cellomics ArrayScan HCS instrument,
[0252] Figure 9 shows antiviral activity of exemplary compounds against human
coronavirus 0C43. Treatment with increasing concentrations of compound 3,
compound
5, compound 6, and compound 7 of Table 1 resulted in dose-dependent inhibition
of
virus infection (shown as % untreated negative control). Calculated 1050
values are
shown.
[0253] Table 4 shows calculated 1050 values of selected compounds against
human
coronavirus 0C43.
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Table 4. 1050 against 0C43
Compound
IC50 (pM) ....................................................
_________________ 2 9.22
_________________ 3 0.54
4 0.09
.............................................. ..õ
0.04
........ , ......................... t--
6 0.04
7 0.02 _____
[0254] Example 12. In vivo pharmacokinetic and toxicological properties of
compounds.
[0255] The in vivo pharmacokinetic (PK) profile and tolerability/toxicity of
the
compounds described herein are evaluated in order to conduct further
characterization
of their antiviral activity in vivo.
[0256] A reverse-phase, HPLC-MS/MS detection method is used for measuring the
concentration of each compound in mouse plasma. Prior to PK profiling, an
initial oral
and intravenous formulation for each compound is developed using a limited
formulation
component screen that is largely focused on maximizing aqueous solubility and
stability
over a small number of storage conditions. Any of the analytical methods as
are known
in the art can be used to measure formulation performance. A formulation is
developed
for each compound following a three tiered strategy:
= Tier 1: pH (pH 3 to 9), buffer, and osmolality adjustment
= Tier 2: addition of ethanol (<10%), propylene glycol (<40%), or
polyethylene glycol
(PEG) 300 or 400 (<60%) co-solvents to enhance solubility
= Tier 3: addition of N-N-dimethylacetamide (DMA, <30%), N-methyl-2-
pyrrolidone
(NMP, <20%), and/or dimethyl sulfoxide (DMSO, <20%) co-solvents or the
cyclodextrins (<40%) as needed to further improve solubility.
[0257] Example 13. In vivo antiviral activity of compounds.
[0258] For compounds that demonstrate adequate performance in in vitro
antiviral,
mechanistic, ADME, and toxicology studies, a preliminary mouse PK study is
performed. Each compound is administered as a single dose to animals by oral
gavage
(<10 ml/kg) or i.v. bolus injection (<5 ml/kg) after an overnight fast.
Multiple animals are
dosed for each dosing group such that 3 animals can be sampled at each time
point.
Blood samples are collected by retro-orbital sinus prior to dosing and at 5,
15, and 30
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minutes, and 1, 2, 4, 8, and 24 hours post-dosing. Drug concentrations are
measured
according to the previously developed bioanalytical method. PK parameters are
evaluated using the WinNonlin software.
[0259] Based upon performance in exploratory PK studies, compounds are further
evaluated for preliminary tolerability and toxicity in mice prior to their
characterization in
antiviral models. Tolerability studies are performed in two stages: an initial
dose
escalation stage (up to 5 doses, each separated by a 5-day washout period) to
determine the maximum tolerable dose (MTD, Phase 1), followed by seven daily
administrations of the MTD to evaluate acute toxicity (Stage 2). All doses are
administered by oral gavage. In an example experiment, five animals of each
sex are
placed on-study in stage 1 and 15 animals per sex per dosing group in Stage 2.
Study
endpoints include a determination of the MTD, physical examination, clinical
observations, hematology, serum chemistry and animal bodyweights. Gross
pathology
is performed on all animals whether found dead, euthanized in extremis, or at
the
intended conclusion of the experiment. The toxicology studies are primarily
exploratory
in nature and intended to identify early toxicological endpoints, and drive
selection of
lead candidates for antiviral animal models.
[0260] Example methods to complete the PK and tolerability studies described
above
are shown in Table 5. These methods may be modified and/or adapted such as a
different route of administration, in order to more accurately measure the
pharmacological properties of a compound.
Table 5 _________________________________
Route of
Study Experimental design istration Outcomes
admin
Single dose Oral bioavailability,
Mouse PK IV and Oral
pharniacokinetic study Cmax, Cl, Vd, AUC0-24,0-
co
Phase 1:
ascending dose tolerability MTD, acute toxicity,
Mouse and MTD determination; Oral hematology, serum
tolerability Phase 2: chemistry, gross
placebo controlled 7-day
pathology
............. toxicity at MTD
[0261] Figure 10 shows results from exploratory PK studies. Administration of
compound 3 of Table 1 via oral (PO) or intravenous (IV) route resulted in
detectable
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levels of compound in serum samples obtained up to 250 minutes post treatment
(Figure 10A). At 4 hours post treatment of compound 3 and compound 7 of Table
1,
there was detectable compound in lung tissue (Figure 10B).
[0262] Example compounds of the disclosure that demonstrate desirable PK
properties, tolerability, antiviral potency, and/or innate immune activating
activity, are
selected for further evaluation in preclinical mouse models of infection.
[0263] Incorporated in the design of these experiments is the determination of
an
effective dose for 50% and 90% suppression of serum viral load (EC50 and EC90)
by
each compound after a standard challenge of virus; for example, 100 pfu of WNV-
TX or
1,000 pfu of influenza virus. Virus quantification in serum and/or target
tissues are
determined by established assay methods including: plaque assay, TCID50 assay,
focus forming assay, viral protein quantification such as through HA assay or
BCA
assay, viral RNA quantification such as through qPCR, and/or antigen
quantification
such as through ELISA.
[0264] The compound actions are tested in virus challenge studies at a minimum
of 2
dose levels including the determined EC50 and EC90 to evaluate their ability
to limit
viral pathogenesis, virus replication, and virus spread. Mice are monitored
for morbidity
and mortality over a range of challenge doses (for example, 10 to 1,000 pfu of
virus)
either alone or in combination with compound treatment beginning 12 hours
before or
24 hours after infection and continuing daily subject to the determined plasma
half-life of
the drug. Compound dose-response analysis and infection time course studies
are also
conducted to evaluate compound efficacy to: 1) limit serum viral load, 2)
limit virus
replication and spread in target organs, and 3) protect against viral
pathogenesis.
[0265] Studies to define effective dosage of drug in vivo and established
mouse virus
infection models are described in Table 6, although this list is not intended
to be
complete and the compounds can be tested in any mouse model for potency
against
any virus infection.
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Table 6
Study Experimental Analysis
Outcomes
design ......................
,
Compound dose Drug measured in
Drug concentration in Define in vivo
determination blood at .?_3 dose
blood; HPLC reverse
levels; 2, 8, 24 hours compound exposure
post treatment p hase
Effective compoundViral burden analysis in .
Viral burden analysis Define In vivo EC50
dose determination serum and/or target
at .?.3 dose levelsand EC90
____________________________________ tissues
Viral pathogenesis Time to moribund
Treatment at defined Define compound
study 1: state, clinical scoring
doses of EC50 and action toward
limiting
EC50 and EC90 for pathologic signs of .
ECK viral pathogenesis
Treatment ___________________________ infection
Viral pathogenesis I
Define compound
study 2: Treatment at defined Viral burden analysis in
action toward limiting
EC50 and EC90 doses of EC50 and serum and various
virus replication and
treatment and time EC90 target organs
spread
course analysis
Mouse WNV Intracranial injection Time to moribund
Define compound
neuroinvasion of WNV-TX; drug state, clinical scoring action toward
limiting
Model treatment at 2 doses for pathologic signs of viral
pathogenesis in
w/ placebo infection _________ the CNS
Mouse Influenza Mortality, viral titer in
Intranasal or tracheal
Model serum/target organs,
instillation of Define compound --1
body temp., clinical
A/PR/8/34, action toward limiting ,
observations,
ANVSN/33 or ht viral pathogenesis,
,
A/Udorn/72; drug virus replication,
and
treatment at 2 doses levels, ywegene cyto kine
spread
expression, markers of
w/ placebo
____________________________________ inflammation __
, ___________________________________________________________________________
,
Mouse RSV Model Mortality, viral titer in
serum/target organs, .
Intranasal or tracheal
body temp., clinical Define compound
instillation of RSV A2 action toward
limiting
observations,
ytokine
Long strain; drug e viral pathogenesis,
bodywight, c
treatment at 2 doses virus replication, and
levels, gene
w/ placebo control spread
expression, markers of
inflammation
................ - ___________
Mouse DNV Model Mortality, viral titer in
serum/target organs,
Define compound
body temp., clinical
IP injection of DV-2; action toward limiting
ry
obseations,
drug treatment at 2 viral pathogenesis,
bodyweight, cytokine
doses w/ placebo virus replication, and
levels, gene
spread
expression, markers of
.................................... inflammation
Mouse MHV-1 Intranasal instillation I Mortality, viral titer in
Define compound
Model of MHV-1; drug serum/target organs, action toward
limiting
treatment at 2 doses body temp., clinical viral
pathognesis,
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-õ
w/ placebo observations, virus replication,
and
bodyweight, cytokine spread
levels, gene
expression, markers of
inflammation
[0266] Mouse WNV model. Efficacy of compounds against WNV can be assayed after
subcutaneous or intracranial (neuroinvasion) infection of virus. Compounds are
administered daily by oral gavage or IP administration over the entire course
of infection
at 2 dose levels plus a placebo control group. Animals are evaluated for study
endpoints
including daily clinical observations, mortality, body weight, and body
temperature. Virus
titer is measured in serum, lymph nodes, spleen, and/or brain. Gene and
cytokine
expression at various time points during infection in compound-treated versus
control
animals can be assayed.
[0267] Mouse influenza model. Virus infection is done by non-surgical
intranasal or
tracheal instillation of influenza virus strains ANVSN/33 and A/Udorn/72.
These
influenza virus strains are two different subtypes (H1 N1 and H3N2) and
exhibit varying
pathogenic properties and clinical presentations in C57BI/6 mice. Compounds
are
administered daily by oral gavage or IP administration over the entire course
of infection
(.?.. 2 weeks) at 2 dose levels plus a placebo control group. Animals are
evaluated for
study endpoints including daily clinical observations, mortality, body weight,
and body
temperature. Virus titer is measured in serum, heart, lung, kidney, liver,
and/or brain.
Gene and cytokine expression at various time points during infection in
compound-
treated versus control animals can be assayed.
[0268] Mouse RSV model. Virus infection is done by non-surgical intranasal or
tracheal
instillation of RSV A2 long strain at a dose that does not cause cytopathic
effects.
Compounds are administered daily by oral gavage or IP administration for 3
weeks, at
2 dose levels or a placebo control. Animals are evaluated for study endpoints
including
daily clinical observations, mortality, body weight, and body temperature.
Virus titer is
measured in serum, blood, and/or lung. Gene and cytokine expression, and
increased
immune cell population counts can be assayed.
[0269] Mouse DNV model. Virus infection is done by intraperitoneal injection
of DNV
type 2 strain. Compounds are administered daily by oral gavage or IP
administration
over the entire course of infection at 2 dose levels plus a placebo control
group. Animals
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are evaluated for study endpoints including daily clinical observations,
mortality, body
weight, and body temperature. Virus titer is measured in serum, blood, heart,
lung,
kidney, liver, and/or brain. Gene and cytokine expression at various time
points during
infection in compound-treated versus control animals can be assayed.
[0270] Mouse hepatitis virus type 1 (MHV-1) coronavirus model. Virus infection
is done
by non-surgical intranasal instillation of MHV-1. Compounds are administered
daily by
oral gavage or IP administration over the entire course of infection (?. 1
week) at 2 dose
levels plus a placebo control group. Animals are evaluated for study endpoints
including
daily clinical observations, mortality, body weight, and body temperature.
Virus titer is
measured in serum, heart, lung, kidney, liver, and/or brain. Gene and cytokine
expression at various time points during infection in compound-treated versus
control
animals can be assayed.
[0271] Figure 11 shows a study performed using the mouse hepatitis virus type
1
(MHV-1) coronavirus model. Treatment with compound 3 of Table 1 resulted in
decreased pathological symptoms including weight loss (A) and increased
survival (B)
after lethal challenge with MHV-1. (C) Virus was decreased in the lung of
animals
treated with compound 3.
[0272] The antiviral activity of example compounds are described in Table 7.
Table 7
Compound DNV2 DNV4 FLU RSV HCOV
ID EC50 EC50 EC50 EC50 EC50
(uM) (uM) (uM) (uM) (uM)
1 >10 2.1 >10 >10 NA
2 6.2 = >5 2 >10 9.2
3 1.9 1.22 ,6 2.3 0.54
4 3.6 NA 1.3 2.8 NA
0.5 5 >10 NA - 0.04
6 2.5 2.8 1.1 1.7 0.04
7 0.71 3.4 NA i. >3 0.02
8 1.2 NA NA NA NA
9 1.8 0.9 1 1,1 1.8 NA
1.4 5.6 3.2 2.9 NA
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_____________________________________________ , ...
11 ----------------------- j 0.5 5.2 NA 1 NA NA
= --------------------------- 12 ------------------------- 0.2 ¨ 2.8 , 6.8 1
7.25 NA
1
................... 13 ____ 2.0 5.5 5.3 14.00 NA
15 0.4 4.6 6.8 >3 = NA
----------------------- = ..
16 NA NA NA NA NA
17 0.5 4.7 4.1 NA NA
19 0.5 0.6 1 .. NA 1-1.2 __ NA
1 ...............................
[0273] Example 14. In vivo adjuvant activity
[0274] To characterize the breadth of adjuvant activity of compounds of the
disclosure,
animal models of vaccination and vaccination plus protection are used. The
studies
include priming animals including rats and mice with compound alone or in
combination
with an antigen and then assessing the adjuvant effect.
[0275] Adjuvant effect is measured by assays for modified, enhanced immune
humoral
and cellular responses. Humoral responses are assessed over time at discrete
times
post vaccination and/or boosting by collecting blood for sera and determining
relative
concentrations of antibody classes (IgM, IgG, IgA or IgE) and/or isotypes
including
IgG1 , IgG2a, IgG2b, IgG2c, IgG3 for IgG antibodies. Moreover, affinity and
avidity of the
generated antibodies is also determined. In instances in which the vaccine
preparation
includes a combination of compound and antigen, the neutralizing activity of
the
generated antibodies is also determined.
[0276] Cellular mediated immune responses induced by the compounds are
measured
by established methods in the field including ex vivo stimulation of
peripheral blood
mononuclear cells, lymph nodes, splenocytes or other secondary lymphoid organs
with
the antigen and measurement of cytokine or chemokine production in the
supernatant at
several times thereafter. Cytokines measured include Th1 type of cytokines
including
IFN gamma and TNF alpha, Th2 type cytokines including IL-4, IL-10, IL-5 and IL-
13 and
Th17 cytokines including IL-17, 1L-21 and IL-23. Chemokines elicited by the
compounds
are also measured including RANTES, 1P-10, MIP1a, MIP1b, and IL-8. T cell
antigen
specific production of cytokines can also be measured by intracellular
cytokine staining
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with fluorescently labeled specific antibodies and flow cytometry or by
ELISPOT. Both
CD4+ ad CD8+ T cell populations are studied.
[0277] Measurement of adjuvant activity at the cellular level is also
determined by
immunophenotyping of surface markers of activation by flow cytometry. CD8 T
cell
antigen-specific responses are also evaluated by intracellular cytokine
staining of
perforin, cell surface marker expression or proliferation assays including
thymidine
incorporation.
[0278] These experiments are designed to validate compound adjuvant activity
in
different combinations of prime-boost schemes and assess how compound-induced
effects on innate immune antiviral programs shape the adaptive immune
responses
mounted to the antigen in the vaccine preparations.
[0279] Detailed immune response analyses of each compound as described above
are
conducted with each selected antigen to determine the immune correlates for
that
particular antigen(s) and compound formulation. These results guide the
protection
studies in which animals vaccinated and boosted with combinations of select
optimized
compounds and desired antigen(s) formulations from select infectious agents
are later
challenged with doses of infectious agent that are known to result in disease
or death of
the animal. Protection afforded by vaccination is typically measured by
monitoring of
clinical symptoms and survival.
[0280] Example 15. Anti-viral activity of compound 12 of Table 1 against Ebola
virus.
[0281] The in vitro efficacy of compound 12 of Table 1 was tested against
Ebola virus
(EBOV). As shown in Figure 12, compound 12 showed greater than a 2 log
reduction in
EBOC titer in vitro. Control titer (pfu/mL) was over 5, whereas the test titer
using
compound 12 was less than 3.5.
[0282] Example 16. Anti-viral effect of compound 8 of Table 1.
[0283] Figure 13 shows the dose response activity of compound 8 of Table 1
against
DENV-2, as FFU/ml.
[0284] EXAMPLE EMBODIMENTS
[0285] 1. A compound represented by the formula
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(R4),,
0
/
H
H
wherein R4 is Rd, SO2Rd, C(=0)Rd, NH C(=0)Rd, Re, OW, or CF3, wherein IR is H
or Ci-
Cio hydrocarbyl, Rd is substituted heterocyclic, unsubstituted heterocyclic,
or
unsubstituted carbocyclic, and Re is substituted heteroaryl or substituted
phenyl; and
n is 1 or 2.
[0286] 2. The compound of embodiment 1, represented by the formula
H
o __________________________________________ 1111
)
H \ /
H
wherein R4 is:
(i) C(=0)Rd and Rd is a pyrrolidonyl group,
OD SO2Rd and Rd is a piperidinyl group,
(iii) NHC(=0)Rd and Rd is a phenyl group or a furanyl group,
(iv) an imidazolyl group, or
(v) a thiazolyl group.
[0287] 3. The compound of embodiment 1, represented by the formula
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II
X H
N ____________________________________ 0
S
4111 Ni>--- HN -.---j 1111411'
H
H
wherein X is NH or O.
028133 4. The compound of embodiment I, wherein R4 is CF3, 011c, or a phenyl
group
Substituted by at least one OCH3 group.
0289i 5. A compound of claim 1 represented by the formula:
0 0 1 \
N is r- ........ \ fq
, \\ / \ Li
.i
-===/- -µ...1---"- \ I) 'K\ ilif
0 \--N
I .// k
.....õ.õ,......,..õ.....k ,,H
,
0 0 it; =:,.\
õ,/ si
./\,,t,= - i .......\\ , s
INN,
= .,,..: , .. == .. I P = /
)\*.,.õ...,<:! >,õõõõ../
i , \, r , ,s i
<,,, .1
µ,,,....-, ,./0.........
\\;..- N
0
..,....,
....e -..;.,. 1(1.%
H 0
\ I =-==l
N'I.'" , 7 s
li
ö
:
84
(9Z 'THIN) JAMS LIIIIISHfIS
S8
HO
rl¨<s 0 ..**VOH
0
OH
o
zl
N
0
I /
101
0
0
= rLe
0
HN
¨</N
ci '1-1
HN 110
I 0
N NAO
tioocosiozsatipd 660ZLI/SIOZ OM
VO-TT-9TOZ 8LT8V6Z0 VD
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/N
0 0
.. loH
s_ Nil)
N
,
0 CH
H3C
S 0 0
N
0 N¨ I __________________________________________________
H.
, or
S
H3C¨(. 1
S
N ) 410
ij
O N
H
[0290] 6. A compound represented by the formula
xi
2 W
Y ' 1
I) ---L- V- Q- Ri
y,4 i
wherein L is NR2, 0, S, C(=O)N, CR2R3CR2R3, CR2R3NR2, CR4=CR4, CR2R30,
CR2R3S, NR2CR2R3, NR2C(=0), NS(0)t, OCR2R3, SCR2R3;
V is (CR2R3)u, C(=0)CR2R3, CR2R30, CR2R3OCR2R3,CR4=CR4, CC, C(=NR2), or
C(=0);
Q is NR2, 0, S(0)t, or a bond;
t= 0, 1, 2; u= 0-3;
wherein a dashed line indicates the presence or absence of a bond;
86
SUBSTITUTE SHEET (RULE 26)
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R1 is Ra, 0R2, or NR2R3;
each Ra is independently H, optionally substituted hydrocarbyl, optionally
substituted
aryl, optionally substituted heteroaryl;
R2 and R3 are each independently Ra, C(=0)Ra, SO2Ra, or R2 and R3 form an
optionally
substituted carbocyclic, heterocarbocyclic, aryl, or heteroaryl ring;
each R4 is independently R2, 0R8, C(=0)Ra, C(=0)NR2R3, NR2R3, NRb(=0)Ra, SRa,
SORa, SO2Ra, SO2NHRa, SO2NR2R3, NCORa, halogen, trihalomethyl, CN, S=0, nitro,
or
two R4 groups form an optionally substituted carbocyclic, heterocarbocyclic,
aryl, or
heteroaryl ring;
W and X are each independently N, NRa, NR5, 0, S, CR2R4 or CR4;
R5 is Ra, C(=0)Ra, SO2Ra, or is not present;
yl, )12, y3 and y4 are each independently CR4 or N; and
NR2R3 may form an optionally substituted heterocylic or heteroaryl ring
including
pyrrolidine, piperidine, morpholine, and piperazine,
[0291] 7. A compound of embodiment 6, the compound having a structure
represented by Formula 1A or 1C
R4
R4 W ,V¨R1
R4-?'
R4
Formula 1A
R4
R4
W
IN _________________________________________ V __ R
41-
\
R4
Formula 1C
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wherein in each of Formula 1A and Formula 1C,
R1 is Ra, 0R2, or NR2R3;
each Ra is independently H, optionally substituted hydrocarbyl, optionally
substituted
aryl, optionally substituted heteroaryl;
R2 and R3 are each independently Ra, C(=0)Ra, or SO2Ra;
each R4 is independently R2, ORa, NR2R3, SRa, SORa, SO2Ra, SO2NHRa, NCORa,
C(=0)Ra, CONR2R3, halogen, trihalomethyl, CN, S=0, or nitro;
V is CR2R3, C(=0), C(=0)CR2R3, or C(=N)R2; and
WisOorS.
[0292] 8. A compound of embodiment 6, the compound having a structure
represented by the Formula 1B
R6
h
S'
I I-- ______ V ___ R'
4- === H
R
R4
Formula 1B
wherein R1 is Ra, 0R2 or NR2R3;
each Ra is independently H, optionally substituted hydrocarbyl, optionally
substituted
aryl, optionally substituted heteroaryl;
R2 and R3 are each independently Ra, C(=0)Ra, or SO2Ra;
each R4 is independently R2, ORa, NR2R3, SRa, SORa, SO2Ra, SO2NHRa, NCORa,
C(=0)Ra, CONR2R3, halogen, trihalomethyl, CN, S=0, or nitro;
R6 is H or CH3,
V is CR2R3, C(=0), C(=0)CR2R3, or C=NR2; and
W is 0 or S.
[0293] 9. A compound of embodiment 7 or 8 wherein R4 is H; and V is C=O.
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[0294] 10. A compound of any one of embodiments 7, 8, or 9 wherein R1 is
optionally substituted phenyl or optionally substituted naphthyl.
[0295] 11. A compound of any one of embodiments 6-10, wherein W is S and X is
N.
[0296] 12. A compound of any one of embodiments 6-10, wherein W is 0 and X is
N.
[0297] 13. A compound of any one of embodiments 6-11 represented by the
formula
R6
\17----= N
i (3
\
S
¨R
...\\,, =-S,
i 11/
>-----
4 =."`""- -"--NI
R \R5
R4
wherein R1 is a phenyl group substituted by at least one halogen, a phenyl
group
substituted by NR2R3, a phenyl group substituted by SO2NR2R3, CR2R3ORd, an
unsubstituted napthyl group, a napthyl group substituted by 0(CR2R3)nRd,
NRa(CR2R3)nRd, NRa(CR2R3)nNR2R3, a two membered ring structure including a
pyridynyl group and a phenyl group, or a two membered ring structure including
a
phenyl group and a dioxolanyl group;
each Ra is independently H or optionally substituted hydrocarbyl (Ci-Cio);
R2 and R3 are each independently Ra, CORa, (CH2)nO, or SO2Ra;
each R4 is independently Ra
Rd is phenyl or morpholino
R5 is H or CH3;
R6 is H or CH3; and
wherein n is 1, 2, 3, or 4.
[0298] 14. A compound of embodiment 13 represented by the formula
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CH3
N
0
S I 0
ic>
H N
CI
CH3
0
0
H N/ I
0
N
0
CI
N
H N
CI
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N
0
S
4111 40,
0
J.
CH3
N
0
io Br
I
CH3
CH3
N
0
S\
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H
\7"------- N
S rah 0
S \ N ...,-***====,
I
H 1411111 Nir- H I
H .
H
)----.4'---N
S 0
I
s> ,..7 /
H ___________________________ el N I
H el
N
H
1
s...,...,e 0
\ ......../ .....
,or
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H C
3
N
H C
-
.\\
S N 0
[0299] 15. A compound of any one of embodiments 5-9 and 11 represented by the
formula
R6
%
1\1/\ 5
R4
wherein R1 is a phenyl group substituted by at least one halogen, a phenyl
group
substituted by NR2R3, a phenyl group substituted by SO2Rd, a napthyl group
optionally
substituted by 0(CR2R3)nRd, or an unsubstituted napthyl group,
each Ra is independently H or optionally substituted Ci-Cio hydrocarbyl;
R2, R3 and each R4 are independently Ra,
Rd is optionally substituted phenyl or optionally substituted morpholino;
R5 is H or CH3;
R6 is H or CH3, and
wherein n is 1, 2, 3, or 4.
[0300] 16. A compound of embodiment 15, represented by the formula:
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H
8!,....4:
,,..,.,..._
-\\,-- 1
0
\\.1/4 ....................
I ;),>: _____________ -N/H
\
-....._ ;':
,
H .1
::........._,õ
,
'*µ',:=;',,\ .
Ft .1
H
3, ...,,..õ...,,:õ...,..r4
- -----,-¨ . : s=,:=
:.' :0
:........... ..:
----'\. ________________________________________ ,
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CH3
0
"'N4' CH3
0
H3C. 11 0, eNC\x,
/ s'
1
= or
= 0,
= \
0 .
4?;
s ,4;"
[0301] 17. A pharmaceutical composition comprising a compound of any one of
embodiments 1 to 16.
[0302] 18. A method of treating a viral infection in a subject comprising
administering
to the subject a therapeutically effective amount of a pharmaceutical
composition of
embodiment 17 thereby treating the viral infection in the subject.
[0303] 19. A method of preventing a viral infection in a subject comprising
administering to the subject a therapeutically effective amount of a
pharmaceutical
composition of embodiment 17.
[0304] 20. A method of embodiment 18 or embodiment 19 wherein the viral
infection
is caused by a virus from one or more of the following families: Arenaviridae,
Arterivirus,
Astroviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae,
Closteroviridae,
Comoviridae, Coronaviridae, Cystoviridae, Filoviridae, Flaviviridae,
Flexiviridae,
Hepadnaviridae, Hepevirus, Herpesviridae, Leviviridae, Luteoviridae,
Mesoniviridae,
Mononegavirales, Mosaic Viruses, Nidovirales, Nodaviridae, Orthomyxoviridae,
Papillomaviridae, Paramyxoviridae, Picobirnaviridae, Picobirnavirus,
Picornaviridae,
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Potyviridae, Reoviridae, Retroviridae, Ron iviridae,
Sequiviridae, Tenuivirus,
Togaviridae, Tombusviridae, Totiviridae, and Tymoviridae.
[0305] 21. A method of embodiment 18 or embodiment 19 wherein the viral
infection
is caused by Alfuy virus, Banzi virus, bovine diarrhea virus, Chikungunya
virus, Dengue
virus (DNV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), human
cytomegalovirus
(hCMV), human immunodeficiency virus (HIV), Ilheus virus, influenza virus
(including
avian and swine isolates), rhinovirus, norovirus, adenovirus, Japanese
encephalitis
virus, Kokobera virus, Kunjin virus, Kyasanur forest disease virus, louping-
ill virus,
measles virus, MERS-coronavirus (MERS), metapneumovirus, any of the Mosaic
Viruses, Murray Valley virus, parainfluenza virus, poliovirus, Powassan virus,
respiratory
syncytial virus (RSV), Rocio virus, SARS-coronavirus (SARS), St. Louis
encephalitis
virus, tick-borne encephalitis virus, West Nile virus (WNV), Ebola virus,
Nipah virus,
Lassa virus, Tacaribe virus, Junin virus, or yellow fever virus.
[0306] 22. A pharmaceutical composition of embodiment 17, for use in therapy.
[0307] 23. A pharmaceutical composition for use according to embodiment 22,
wherein said pharmaceutical composition is administered as an adjuvant for a
prophylactic or therapeutic vaccine.
[0308] 24. A method of modulating the innate immune response in a eukaryotic
cell,
comprising administering to the cell a compound of any one of embodiments 1 to
16.
[0309] 25. A method of embodiment 24, wherein the cell is in vivo.
[0310] 26. A method of embodiment 25, wherein the cell is in vitro.
[0311] 27. A method of treating a viral infection in a subject comprising
administering
to the subject a therapeutically effective amount of a pharmaceutical
composition having
the structure
0 \
/V ¨1
N
and wherein the viral infection is caused by Ebola virus.
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[0312] As will be understood by one of ordinary skill in the art, each
embodiment
disclosed herein can comprise, consist essentially of or consist of its
particular stated
element, step, ingredient or component. Thus, the terms "include" or
"including" should
be interpreted to recite: "comprise, consist of, or consist essentially of."
As used herein,
the transition term "comprise" or "comprises" means includes, but is not
limited to, and
allows for the inclusion of unspecified elements, steps, ingredients, or
components,
even in major amounts. The transitional phrase "consisting of" excludes any
element,
step, ingredient or component not specified. The transition phrase "consisting
essentially of" limits the scope of the embodiment to the specified elements,
steps,
ingredients or components and to those that do not materially affect the
embodiment.
As used herein, a material effect would cause a statistically significant
reduction in a
disclosed compound's or composition's ability to treat a viral infection in a
subject;
reduce viral protein in a subject or assay; reduce viral RNA in a subject or
assay or
reduce virus in a cell culture.
[0313] Unless otherwise indicated, all numbers expressing quantities of
ingredients,
properties such as molecular weight, reaction conditions, and so forth used in
the
specification and claims are to be understood as being modified in all
instances by the
term "about," Accordingly, unless indicated to the contrary, the numerical
parameters
set forth in the specification and attached claims are approximations that can
vary
depending upon the desired properties sought to be obtained by the present
disclosure.
At the very least, and not as an attempt to limit the application of the
doctrine of
equivalents to the scope of the claims, each numerical parameter should at
least be
construed in light of the number of reported significant digits and by
applying ordinary
rounding techniques. When further clarity is required, the term "about" has
the meaning
reasonably ascribed to it by a person skilled in the art when used in
conjunction with a
stated numerical value or range, i.e. denoting somewhat more or somewhat less
than
the stated value or range, to within a range of 20% of the stated value; 19%
of the
stated value; 18% of the stated value; 17% of the stated value; 16% of the
stated
value; 15% of the stated value; 14% of the stated value; 13% of the stated
value;
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12% of the stated value; 11% of the stated value; 10% of the stated value;
9% of
the stated value; 8% of the stated value; 7% of the stated value; 6% of the
stated
value; 5% of the stated value; 4% of the stated value; 3% of the stated
value; 2%
of the stated value; or 1% of the stated value.
[0314] Notwithstanding that the numerical ranges and parameters setting forth
the
broad scope of the disclosure are approximations, the numerical values set
forth in the
specific examples are reported as precisely as possible. Any numerical value,
however,
inherently contains certain errors necessarily resulting from the standard
deviation
found in their respective testing measurements_
[0315] The terms "a," "an," "the" and similar referents used in the context of
describing
the disclosure (especially in the context of the following claims) are to be
construed to
cover both the singular and the plural, unless otherwise indicated herein or
clearly
contradicted by context. Recitation of ranges of values herein is merely
intended to
serve as a shorthand method of referring individually to each separate value
falling
within the range. Unless otherwise indicated herein, each individual value is
incorporated into the specification as if it were individually recited herein_
All methods
described herein can be performed in any suitable order unless otherwise
indicated
herein or otherwise clearly contradicted by context. The use of any and all
examples, or
exemplary language (e.g., "such as") provided herein is intended merely to
better
illuminate the disclosure and does not pose a limitation on the scope of the
disclosure
otherwise claimed. No language in the specification should be construed as
indicating
any non-claimed element essential to the practice of the disclosure.
[0316] Groupings of alternative elements or embodiments of the disclosure
disclosed
herein are not to be construed as limitations. Each group member can be
referred to
and claimed individually or in any combination with other members of the group
or other
elements found herein. It is anticipated that one or more members of a group
can be
included in, or deleted from, a group for reasons of convenience and/or
patentability.
When any such inclusion or deletion occurs, the specification is deemed to
contain the
group as modified thus fulfilling the written description of all Markush
groups used in the
appended claims.
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[0317] Certain embodiments of this disclosure are described herein, including
the best
mode known to the inventors for carrying out the disclosure. Of course,
variations on
these described embodiments will become apparent to those of ordinary skill in
the art
upon reading the foregoing description. The inventor expects skilled artisans
to employ
such variations as appropriate, and the inventors intend for the disclosure to
be
practiced otherwise than specifically described herein. Accordingly, this
disclosure
includes all modifications and equivalents of the subject matter recited in
the claims
appended hereto as permitted by applicable law. Moreover, any combination of
the
above-described elements in all possible variations thereof is encompassed by
the
disclosure unless otherwise indicated herein or otherwise clearly contradicted
by
context.
[0318] Numerous references have been made to publications, patents and/or
patent
applications (collectively "references") throughout this specification. Each
of the cited
references is individually incorporated herein by reference for their
particular cited
teachings.
[0319] In closing, it is to be understood that the embodiments of the
disclosure
disclosed herein are illustrative of the principles of the present disclosure.
Other
modifications that may be employed are within the scope of the disclosure.
Thus, by
way of example, but not of limitation, alternative configurations of the
present disclosure
may be utilized in accordance with the teachings herein. Accordingly, the
present
disclosure is not limited to that precisely as shown and described.
99
